Preparation and uses of reactive oxygen species scavenger derivatives

ABSTRACT

including certain quinone derivatives, and the corresponding pharmaceutical compositions, which may serve to modulate ferroptosis in a subject. Also disclosed herein are the preparations of these compounds and pharmaceutical compositions and their potential uses in the manufacture of a medicament in reducing reactive oxygen species (ROS) in a cell and for preventing, treating, ameliorating certain related disorder or a disease.

This application is a divisional of U.S. application Ser. No. 16/786,369, filed on Feb. 10, 2020, now allowed, which claims the benefit under 35 U.S.C. § 111(a) of PCT International Application No. PCT/CN2017/097805 filed on Aug. 17, 2017, which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides compositions and methods useful for treatment or suppression of diseases, developmental delays and symptoms related to oxidative stress. More specifically, the compositions and methods comprise administrating an effective amount of a compound that is a ferroptosis modulator.

BACKGROUND OF INVENTION

While localized ROS (reactive oxygen species) are critical to the eukaryotic cell's biological metabolism and development, excessive flux of unrestrained ROS leads to cellular dysfunctions and/or death.

Since its introduction in 2012, ferroptosis has emerged as a key mechanistic pathway for excess ROS flux (especially in the mitochondria) which ultimately leads to non-apoptotic cell death. As the key element in ferroptosis, the loss of glutathione peroxidase 4 (GPx4) activity allows for a lethal excess of ROS which then leads to cellular destruction by oxidative stress. Since GPx4 uses glutathione (GSH) as the key co-factor for scavenging ROS, GSH production deficit can arise from blocked induction (e.g., via known small compounds such as erastin and excess glutamates) of its ‘up-stream’ Xc system (a glutamate/cystine anti-transporter). On the other hand, inducing excess ROS flux via ferroptosis can kill certain cancer cells.

Based on the above reviews as well as references about some other inhibitors attributable to their anti-ferroptotic mechanisms of action (e.g., ferrostatins: patent applications-US 2016/0297748 and WO 2013/152039, quinones such as MitoQ, small peptides such as SS-31: (https://www.ncbi.nlm nih.gov/pmc/articles/PMC4267688/accessed Jul. 7, 2017 and patent publication WO2004070054 (A2)); nitroxides such as XJB-5-131 and JP4-039 analogs (patent publication WO2012112851 (A2)). XJB-5-131 has also been applied in the application of inhibition of ferroptosis, wherein it plays a critical role of intramitochondrial lipid peroxidation in ferroptosis. Also, the reference compound ferrostatin-1 and XJB-5-131 may share similar mechanisms of action or that both compounds operate on the same signaling pathway. Ferroptosis can be linked to a variety of well-known diseases broadly grouped as involving the non-central nervous system (non-CNS) or CNS.

Non-CNS: cardiovascular (e.g., atherosclerosis, hypertensive cardiomyopathy, congestive heart failure, stroke, etc.), metabolic (e.g., diabetes and its related complications such as neuropathy, hyperlipidemia, etc.), urinary (e.g., acute kidney injury (AKI)), age related diseases (e.g., skeletal muscle atrophy, and dry aged macular degeneration (Dry-AMD) and trauma (e.g., radiation injury, inducing rhabdomyolysis, traumatic brain injury, major surgery, etc.). In the case of AKI, US FDA today has yet to approve a drug to specifically cure this disease which especially affects ˜30% of US patients hospitalized in intensive care units. When AKI unfortunately progresses to the late/chronic stages, hemo-dialysis and ultimately kidney replacement become the major remaining treatment options.

CNS: neuro-motor (e.g., Parkinson's, Huntington's, amyotrophic lateral sclerosis, epilepsy, etc.) and cognitive (e.g., Alzheimer's). In the case of epilepsy which affects over 50 million people globally, a significant minority (20-30%) of patients are/become resistant to more than 20 currently approved drugs. Undoubtedly, ongoing research will likely link more diseases due to and more inhibitors of ferroptosis since its 2012 introduction. Thus as exemplified by AKI and epilepsy some other diseases to be linked to ferroptosis in the future also urgently need better (likely novel) drugs.

SUMMARY OF THE INVENTION

The following is only an overview of some aspects of the present invention, but is not limited thereto. All references of this specification are incorporated herein by reference in their entirety. When the disclosure of this specification is different with citations, the disclosure of this specification shall prevail. The present invention provides compounds and pharmaceutical compositions, which modulates ferroptosis in a subject; include certain quinone derivatives, their preparation, and the corresponding pharmaceutical compositions. The compounds and/or pharmaceutical compositions of the present invention can be potentially used in the manufacture of a medicament for preventing, treating, ameliorating certain disorder or a disease in a patient.

One aspect of the present invention is the provision of a compound of the following Formula (I)a or (I)b, pharmaceutically acceptable salts and individual enantiomers or diastereomers thereof.

Wherein z is 0 or 1;

-   -   L is absent, or is selected from a group consisting of: C1-C10         alkyl, C1-C10 alkoxyl, cycloalkyl, C1-C10 alkylcycloalkyl,         heterocycloalkyl, C1-C10 alkylheterocycloalkyl, aryl, C1-C10         alkylaryl, heteroaryl, and C1-C10 alkylheteroaryl, wherein said         cycloalkyl, or heterocycloalkyl has about 3 to about 7 ring         carbons, and said aryl or heteroaryl has about 5 to about 10         ring carbons;         Q is selected from a group consisting of:

-   -   wherein n is an integer selected from 0 to 10;         -   R₁ or R₂ is H or CH₃;         -   R₃ or R₄ is H or C1-C5alkyl;         -   W is absent, 0 or S; and         -   i) when W is absent, R₃ and R₄ can, optionally, together             form the following structure:

-   -   -   ii) when W is O, OR₃ and OR₄ can, optionally, together form             the following structure:

PEP is a peptidyl moiety having the following structure:

wherein a * mark denotes a chiral center in either an (S) or (R) configuration and m is an integer selected from 0 to 10;

X₁ is selected from a group consisting of H, —(C═O)—O-Rm, —(SO)—O-Rm, —(SO₂)—O-Rm, —(SO₂)—N-(Rm)₂, and —(C═O)—N-(Rm)₂, where Rm is C₁-C₆ alkyl, C₁-C₆ alkoxyl, C₁-C₆ alkenyl, C₁-C₆ haloalkyl, cycloalkyl, C₁-C₆ alkylcycloalkyl, heterocycloalkyl, C₁-C₆ alkylheterocycloalkyl, aryl, C₁-C₆ alkylaryl, heteroaryl, and C₁-C₆ alkylheteroaryl, wherein said cycloalkyl, or heterocycloalkyl has about 3 to about 7 ring carbons, and said aryl or heteroaryl has about 5 to about 10 ring carbons;

X₂, X₃ or X₄ is selected from a group consisting of C1-C10 alkyl, C1-C10 alkenyl, aryl, C₁-C₆ alkylaryl, heteroaryl, C₁-C₆ alkylheteroaryl, wherein said aryl or heteroaryl having from 5 to 6 ring carbons;

A is absent or selected from a group consisting of: Ala, Leu, Ile, Phe, Met, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Gln, His, Lys, Arg, Asp, Glu, and Val, each of which can be in either a D or L configuration; wherein the amino residue is either unprotected, or protected by a protecting group selected from a group consisting of Cbz and Fmoc;

G is absent, or selected from a group consisting of: —O—, —S—, —NH—, —NH—(C═O)—O—, —O—(C═O)—NH—, —NH—(C═O)—NH—, —NHSO₂—, —SO—, and —SO₂—.

A further aspect of the present invention is the provision of a compound of the aforementioned Formula (I)a or (I)b, wherein PEP is preferably

wherein a * mark denotes a chiral center that can be either in an (S) or (R) configuration;

-   -   X₁ is selected from a group consisting of H, —(C═O)—O-Rm,         —(SO)—O-Rm, —(SO₂)—O-Rm, —(SO₂)—N-(Rm)₂, and —(C═O)—N-(Rm)₂,         where Rm is C₁-C₆ alkyl, C₁-C₆ alkoxyl, C₁-C₆ alkenyl, C₁-C₆         haloalkyl, cycloalkyl, C₁-C₆ alkylcycloalkyl, heterocycloalkyl,         C₁-C₆ alkylheterocycloalkyl, aryl, C₁-C₆ alkylaryl, heteroaryl,         and C₁-C₆ alkylheteroaryl, wherein said cycloalkyl, or         heterocycloalkyl has about 3 to about 7 ring carbons, and said         aryl or heteroaryl has about 5 to about 10 ring carbons;     -   X₂, X₃ or X₄ is selected from a group consisting of C₁-C₁₀         alkyl, C₁-C₁₀ alkenyl, aryl, C₁-C₆ alkylaryl, heteroaryl, C₁-C₆         alkylheteroaryl, wherein said aryl or heteroaryl having from 5         to 6 ring carbons.     -   A is absent or selected from a group consisting of: Ala, Leu,         Ile, Phe, Met, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Gln, His, Lys,         Arg, Asp, Glu, and Val, each of which can be in either a D or L         configuration; wherein the amino residue is either unprotected,         or protected by a protecting group selected from a group         consisting of Cbz and Fmoc.

In a further aspect, the invention relates to pharmaceutical compositions each comprising an effective amount of at least one compound of Formula (I)a or (I)b or a pharmaceutically acceptable salt of a compound of Formula (I)a or (I)b, and individual enantiomers and diastereomers thereof. Pharmaceutical compositions according to the invention may further comprise at least one pharmaceutically acceptable excipient, carrier, adjuvant, solvent, support or a combination thereof and may further comprise therapeutically effective amounts of one or more, optional, adjunctive active ingredients.

In yet another aspect of the present invention, the compounds of Formula (I)a or (I)b or a pharmaceutically acceptable salt of a compound of Formula (I)a or (I)b, and individual enantiomers and diastereomers thereof, are useful as ferroptosis modulators. Thus, the invention is directed to the use of an effective amount of at least one aforementioned compound of Formula (I)a or (I)b or a pharmaceutically acceptable salt of a compound of Formula (I)a or (I)b, and individual enantiomers and diastereomers thereof in the manufacture of a medicament to be used in a method for reducing reactive oxygen species (ROS) in a cell comprising contacting a cell with an effective amount of said compound as a ferroptosis modulator.

In still another aspect of the present invention, there is directed to a method for modulating ferroptosis in a subject, comprising exposing the subject to an effective amount of at least one compound of Formula (I)a or (I)b, or a pharmaceutically acceptable salt of a compound of Formula (I)a or (I)b, and individual enantiomers and diastereomers thereof, and at least one pharmaceutically acceptable excipient, carrier, adjuvant, solvent, support or a combination thereof, and further therapeutically effective amounts of one or more, optional, adjunctive active ingredients.

Another aspect of the present invention concerns the use of pharmaceutical compositions each comprising an effective amount of at least one compound of Formula (I)a or (I)b or a pharmaceutically acceptable salt of a compound of Formula (I)a or (I)b, and individual enantiomers and diastereomers thereof, and at least one pharmaceutically acceptable excipient, carrier, adjuvant, solvent, support or a combination thereof, and further therapeutically effective amounts of one or more, optional, adjunctive active ingredients, in the manufacture of a medicament for preventing, treating or lessening an oxidative stress related disorder or disease in a patient by administering therapeutically effective amounts of said compound or said pharmaceutical composition as a ferroptosis modulator.

In yet another aspect, the invention is directed to a method for preventing, treating or lessening an oxidative stress related disorder or disease in a patient by modulating certain ferroptosis process in said patient by administering to the patient a therapeutically effective amount of the aforementioned pharmaceutical compositions each comprising an effective amount of at least one compound of Formula (I)a or (I)b or a pharmaceutically acceptable salt of a compound of Formula (I)a or (I)b, and individual enantiomers and diastereomers thereof, and at least one pharmaceutically acceptable excipient, carrier, adjuvant, solvent, support or a combination thereof, and further therapeutically effective amounts of one or more, optional, adjunctive active ingredients.

In yet another aspect, the present invention is directed to methods of making compounds of Formula (I)a or (I)b and pharmaceutically acceptable salts thereof.

In certain embodiments of the compounds, pharmaceutical compositions, and methods of the invention, the compound of Formula (I)a or (I)b is a compound selected from those species described or exemplified in the detailed description below, or is a pharmaceutically acceptable salt of such a compound.

Another preferred embodiment, the present invention is directed to methods of preparing pharmaceutical compositions each comprising an effective amount of at least one compound of Formula (I)a or (I)b, or a pharmaceutically acceptable salt of a compound of Formula (I)a or (I)b, and further individual enantiomers and diastereomers thereof. As aforementioned, pharmaceutical compositions according to the invention may further comprise at least one pharmaceutically acceptable excipient, carrier, adjuvant, solvent, support or a combination thereof and further comprise therapeutically effective amounts of one or more, optional, adjunctive active ingredients.

If formulated as a fixed dose, such combination products employ the compounds of this invention within the dosage range described herein (or as known to those skilled in the art) and the other pharmaceutically active agents or treatments within its dosage range. For example, the CDCl₂ inhibitor olomucine has been found to act synergistically with known cytotoxic agents in inducing apoptosis (J. Cell Sci., (1995) 108, 2897). The compounds of the invention may also be administered sequentially with known anticancer or cytotoxic agents when a combination formulation is inappropriate. In any combination treatment, the invention is not limited in the sequence of administration; compounds of Formula (I)a or (I)b may be administered either prior to or after administration of the known anticancer or cytotoxic agent. For example, the cytotoxic activity of the cyclin-dependent kinase inhibitor flavopiridol is affected by the sequence of administration with anticancer agents (Cancer Research, (1997) 57, 3375). Such techniques are within the skills of persons skilled in the art as well as attending physicians.

Any of the aforementioned methods may be augmented by administration of fluids (such as water), loop diuretics, one or more of a chemotherapeutic or antineoplastic agent, such as leucovorin and fluorouracil, and an adjunctive chemotherapeutic agent (such as filgrastim and erythropoietin), or any combination of the foregoing.

Yet another embodiment is a method for administering a compound of the instant invention to a subject (e.g., a human) in need thereof by administering to the subject the pharmaceutical formulation of the present invention.

Yet another embodiment is a method of preparing a pharmaceutical formulation of the present invention by mixing at least one pharmaceutically acceptable compound of the present invention, and, optionally, one or more pharmaceutically acceptable additives or excipients.

For preparing pharmaceutical compositions from the compounds described by this invention, inert, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, beads, cachets and suppositories. The powders and tablets may be comprised of from about 5 to about 95 percent active ingredient. Suitable solid carriers are known in the art, e.g., magnesium carbonate, magnesium stearate, talc, sugar or lactose. Tablets, powders, cachets and capsules can be used as solid dosage forms suitable for oral administration. Examples of pharmaceutically acceptable carriers and methods of manufacture for various compositions may be found in A. Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition, (1990), Mack Publishing Co., Easton, Pa.

Liquid form preparations include solutions, suspensions and emulsions. As an example may be mentioned water or water-propylene glycol solutions for parenteral injection or addition of sweeteners and opacifiers for oral solutions, suspensions and emulsions. Liquid form preparations may also include solutions for intranasal administration.

Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier, such as an inert compressed gas, e.g., nitrogen.

Also included are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions and emulsions.

The compounds of the invention may also be deliverable transdermally. The transdermal compositions can take the form of creams, lotions, aerosols and/or emulsions and can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.

The compounds of this invention may also be delivered subcutaneously.

Preferably the compound is administered orally or intravenously or via an eye drop or an intravitreous injection

Preferably, the pharmaceutical preparation is in a unit dosage form. In such form, the preparation is subdivided into suitably sized unit doses containing appropriate quantities of the active component, e.g., an effective amount to achieve the desired purpose.

The actual dosage employed may be varied depending upon the requirements of the patient and the severity of the condition being treated. Determination of the proper dosage regimen for a particular situation is within the skill of the art. For convenience, the total daily dosage may be divided and administered in portions during the day as required. The amount and frequency of administration of the compounds of the invention and/or the pharmaceutically acceptable salts thereof will be regulated according to the judgment of the attending clinician considering such factors as age, condition and size of the patient as well as severity of the symptoms being treated.

Any embodiment disclosed herein can be combined with other embodiments as long as they are not contradictory to one another, even though the embodiments are described under different aspects of the invention. In addition, any technical feature in one embodiment can be applied to the corresponding technical feature in other embodiments as long as they are not contradictory to one another, even though the embodiments are described under different aspects of the invention.

The foregoing merely summarizes certain aspects disclosed herein and is not intended to be limiting in nature. These aspects and other aspects and additional embodiments, features, and advantages of the invention will be apparent from the following detailed description and through practice of the invention.

DETAILED DESCRIPTION AND PARTICULAR EMBODIMENTS

For the sake of brevity, the disclosures of the publications cited in this specification, including patents and patent applications, are herein incorporated by reference in their entirety.

Most chemical names were generated using IUPAC nomenclature herein. Some chemical names were generated using different nomenclatures or alternative or commercial names known in the art. In the case of conflict between names and structures, the structures prevail.

Definitions and General Terminology

Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structures and formulas. The invention is intended to cover all alternatives, modifications and equivalents which may be included within the scope of the present invention as defined by the claims. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described herein. In the event that one or more of the incorporated literature, patents, and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.

It is further appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable sub-combination.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as are commonly understood by one skilled in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entirety.

As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, and the Handbook of Chemistry and Physics, 75th Ed. 1994. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry” by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, the entire contents of which are hereby incorporated by reference.

As used above, and throughout this disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings. If a definition is missing, the conventional definition as known to one skilled in the art controls. If a definition provided herein conflicts or is different from a definition provided in any cited publication, the definition provided herein controls.

As used herein, the terms “including”, “containing” and “comprising” are used in their open, non-limiting sense.

As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.

To provide a more concise description, some of the quantitative expressions given herein are not qualified with the term “about”. It is understood that, whether the term “about” is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/or measurement conditions for such given value. Whenever a yield is given as a percentage, such yield refers to a mass of the entity for which the yield is given with respect to the maximum amount of the same entity that could be obtained under the particular stoichiometric conditions. Concentrations that are given as percentages refer to mass ratios, unless indicated differently.

Chemical Definitions

As used herein, “alkyl” refers to a saturated, straight- or branched-chain hydrocarbon group having from 1 to 12 carbon atoms. Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and the like, and longer alkyl groups, such as heptyl, octyl, and the like. As used herein, “lower alkyl” means an alkyl having from 1 to 6 carbon atoms.

The term “alkylamino” as used herein denotes an amino group as defined herein wherein one hydrogen atom of the amino group is replaced by an alkyl group as defined herein Aminoalkyl groups can be defined by the following general formula —NH— alkyl. This general formula includes groups of the following general formulae: —NH—C₁-C₁₀ alkyl and —NH—C₁-C₆ alkyl. Examples of aminoalkyl groups include, but are not limited to aminomethyl, aminoethyl, aminopropyl, aminobutyl.

The term “alkoxy” as used herein includes —O-(alkyl), wherein alkyl is defined above.

As used herein, “alkoxyalkyl” means -(alkylenyl)-O-(alkyl), wherein each “alkyl” is independently an alkyl group defined above.

The term “amine” as used herein refers to an —NH₂ group.

“Aryl” means a mono-, bi-, or tricyclic aromatic group, wherein all rings of the group are aromatic. For bi- or tricyclic systems, the individual aromatic rings are fused to one another. Exemplary aryl groups include, but are not limited to, phenyl, naphthalene, and anthracene.

“Aryloxy” as used herein refers to an —O-(aryl) group, wherein aryl is defined as above.

“Arylalkyl” as used herein refers to an -(alkylenyl)-(aryl) group, wherein alkylenyl and aryl are as defined above. Non-limiting examples of arylalkyls comprise a lower alkyl group. Non-limiting examples of suitable arylalkyl groups include benzyl, 2-phenethyl, and naphthalenylmethyl.

“Arylalkoxy” as used herein refers to an —O-(alkylenyl)-aryl group wherein alkylenyl and aryl are as defined above.

The term “deuterium” “deuterated” as used herein means being, being substituted with, a stable isotope of hydrogen having one proton and one neutron.

The term “halogen” as used herein refers to fluorine, chlorine, bromine, or iodine. The term “halo” represents chloro, fluoro, bromo, or iodo.

The term “haloalkyl” denotes an alkyl group as defined above wherein one or more, for example one, two, or three of the hydrogen atoms of the alkyl group are replaced by a halogen atom, for example fluoro, bromo, or chloro, in particular fluoro. Examples of haloalkyl include, but are not limited to, monofluoro-, difluoro-, or trifluoro-methyl, -ethyl or -propyl, for example, 3,3,3-trifluoropropyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, fluoromethyl, difluoromethyl, or trifluoromethyl, or bromoethyl or chloroethyl. Similarly, the term “fluoroalkyl” refers to an alkyl group as defined above substituted with one or more, for example one, two, or three fluorine atoms.

The term “haloalkoxy” as used herein refers to an —O-(haloalkyl) group wherein haloalkyl is defined as above. Exemplary haloalkoxy groups are bromoethoxy, chloroethoxy, trifluoromethoxy and 2,2,2-trifluoroethoxy.

The term “hydroxy” means an —OH group.

The term “hydroxyalkyl” denotes an alkyl group that is substituted by at least one hydroxy group, for example, one, two or three hydroxy group(s). The alkyl portion of the hydroxyalkyl group provides the connection point to the remainder of a molecule. Examples of hydroxyalkyl groups include, but are not limited to, hydroxymethyl, hydroxyethyl, 1-hydroxypropyl, 2-hydroxyisopropyl, 1,4-dihydroxybutyl, and the like.

The term “oxo” means an ═O group and may be attached to a carbon atom or a sulfur atom. The term “N-oxide” refers to the oxidized form of a nitrogen atom.

As used herein, the term “cycloalkyl” refers to a saturated or partially saturated, monocyclic, fused polycyclic, bridged polycyclic, or spiro polycyclic carbocycle having from 3 to 12 ring carbon atoms. A non-limiting category of cycloalkyl groups are saturated or partially saturated, monocyclic carbocycles having from 3 to 6 carbon atoms. Illustrative examples of cycloalkyl groups include, but are not limited to, the following moieties:

The term “cycloalkoxy” refers to a —O-(cycloalkyl) group.

As used herein, the term “heteroaryl” refers to a monocyclic, or fused polycyclic, aromatic heterocycle having from three to 15 ring atoms that are selected from carbon, oxygen, nitrogen, selenium and sulfur. Suitable heteroaryl groups do not include ring systems that must be charged to be aromatic, such as pyrylium. Some suitable 5-membered heteroaryl rings (as a monocyclic heteroaryl or as part of a polycyclic heteroaryl) have one oxygen, sulfur, or nitrogen atom, or one nitrogen plus one oxygen or sulfur, or 2, 3, or 4 nitrogen atoms. Some suitable 6-membered heteroaryl rings (as a monocyclic heteroaryl or as part of a polycyclic heteroaryl) have 1, 2, or 3 nitrogen atoms. Examples of heteroaryl groups include, but are not limited to, pyridinyl, imidazolyl, imidazopyridinyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, triazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl.

Those skilled in the art will recognize that the species of heteroaryl, and cycloalkyl groups listed or illustrated above are not exhaustive, and that additional species within the scope of these defined terms may also be selected.

As described herein, compounds disclosed herein may optionally be substituted with one or more substituents, or as exemplified by particular classes, subclasses, and species of the invention.

As used herein, the term “substituted” means that the specified group or moiety bears one or more suitable substituents. As used herein, the term “unsubstituted” means that the specified group bears no substituents. As used herein, the term “optionally substituted” means that the specified group is unsubstituted or substituted by the specified number of substituents. Where the term “substituted” is used to describe a structural system, the substitution is meant to occur at any valency-allowed position on the system.

As used herein, the expression “one or more substituents” denotes one to maximum possible number of substitution(s) that can occur at any valency-allowed position on the system. In a certain embodiment, one or more substituent means 1, 2, 3, 4, or 5 substituents. In another embodiment, one or more substituent means 1, 2, or 3 substituents.

Any atom that is represented herein with an unsatisfied valence is assumed to have the sufficient number of hydrogen atoms to satisfy the atom's valence.

When any variable (e.g., alkyl, alkylenyl, heteroaryl, R₁, R₂, or R_(a)) appears in more than one place in any formula or description provided herein, the definition of that variable on each occurrence is independent of its definition at every other occurrence.

Numerical ranges, as used herein, are intended to include sequential whole numbers. For example, a range expressed as “from 0 to 4” or “0-4” includes 0, 1, 2, 3 and 4, while a range expressed as “10-20%” includes 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% and 20%. Similarly, numerical ranges are also intended to include sequential fractional integers. For example, a range expressed as “1-2%” would include 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% and 2.0%.

When a multifunctional moiety is shown, the point of attachment to the core is indicated by a line or hyphen. For example, aryloxy- refers to a moiety in which an oxygen atom is the point of attachment to the core molecule while aryl is attached to the oxygen atom.

Additional Definitions

As used herein, the term “subject” encompasses mammals and non-mammals Examples of mammals include, but are not limited to, any member of the Mammalian class: humans; non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; and laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish and the like. In one embodiment of the present invention, the mammal is a human

“Patient” includes both human and animals

The term “inhibitor” refers to a molecule such as a compound, a drug, an enzyme activator, or a hormone that blocks or otherwise interferes with a particular biologic activity.

The term “modulator” refers to a molecule, such as a compound of the present invention, that increases or decreases, or otherwise affects the activity of a given protein, receptor and/or ion channels.

The terms “effective amount” or “therapeutically effective amount” refer to a sufficient amount of the agent to provide the desired biological result. That result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease or medical condition, or any other desired alteration of a biological system. For example, an “effective amount” for therapeutic use is the amount of a compound, or of a composition comprising the compound, that is required to provide a clinically relevant change in a disease state, symptom, or medical condition. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. Thus, the expression “effective amount” generally refers to the quantity for which the active substance has a therapeutically desired effect.

As used herein, the terms “treat” or “treatment” encompass both “preventative” and “curative” treatment. “Preventative” treatment is meant to indicate a postponement of development of a disease, a symptom of a disease, or medical condition, suppressing symptoms that may appear, or reducing the risk of developing or recurrence of a disease or symptom. “Curative” treatment includes reducing the severity of or suppressing the worsening of an existing disease, symptom, or condition. Thus, treatment includes ameliorating or preventing the worsening of existing disease symptoms, preventing additional symptoms from occurring, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disorder or disease, e.g., arresting the development of the disorder or disease, relieving the disorder or disease, causing regression of the disorder or disease, relieving a condition caused by the disease or disorder, or stopping the symptoms of the disease or disorder.

As used herein, the terms “administration of” and “administering a” compound should be understood to mean providing a compound of the invention, pharmaceutical composition comprising a compound or a prodrug of a compound of the invention to an individual in need thereof. It is recognized that one skilled in the non-limiting art can treat a patient presently afflicted with related diseases or disorders or by prophylactically treat a patient afflicted with the diseases or disorders with an effective amount of the compound of the present invention.

The term “composition” as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts. Such term in relation to pharmaceutical composition, is intended to encompass a product comprising the active ingredient(s) and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from a combination, complexation or aggregation of any two or more of the ingredients, or from the other types of reactions or interactions such as to cause the dissociation of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by mixing a compound of the present invention and a pharmaceutically acceptable carrier.

Additional Chemical Descriptions

Any formula given herein is intended to represent compounds having structures depicted by the structural formula as well as certain variations or forms. For example, compounds of any formula given herein may have asymmetric or chiral centers and therefore exist in different stereoisomeric forms. All stereoisomers, including optical isomers, enantiomers, and diastereomers, of the compounds of the general formula, and mixtures thereof, are considered to fall within the scope of the formula. Furthermore, certain structures may exist as geometric isomers (i.e., cis and trans isomers), as tautomers, or as atropisomers. All such isomeric forms, and mixtures thereof, are contemplated herein as part of the present invention. Thus, any formula given herein is intended to represent a racemate, one or more enantiomeric forms, one or more diastereomeric forms, one or more tautomeric or atropisomeric forms, and mixtures thereof.

“Stereoisomer” refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space. Stereoisomers include enantiomer, diastereomers, conformer (rotamer), geometric (cis/trans) isomer, atropisomer etc. . . .

“Chiral” refers to molecules which have the property of non-superimposability of the mirror image partner, while the term “achiral” refers to molecules which are superimposable on their mirror image partner.

“Enantiomers” refers to two stereoisomers of a compound which are non-superimposable mirror images of one another.

“Diastereomer” refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g., melting points, boiling points, spectral properties or biological activities. A mixture of diastereomers may be separated under high resolution analytical procedures such as electrophoresis and chromatography such as HPLC.

Stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., “Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., New York, 1994.

Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (−) are employed to designate the sign of rotation of plane-polarized light by the compound, with (−) or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. A specific stereoisomer may be referred to as an enantiomer, and a mixture of such stereoisomers is called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process.

Any asymmetric atom (e.g., carbon or the like) of the compound(s) disclosed herein can be in racemic or enantiomerically enriched, for example the (R)-, (S)- or (R, S)-configuration. In certain embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess in the (R)- or (S)-configuration.

Depending on the choice of the starting materials and procedures, the compounds can be present in the form of one of the possible stereoisomers or as mixtures thereof, such as racemates and diastereoisomer mixtures, depending on the number of asymmetric carbon atoms. Optically active (R)- and (S)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If the compound contains a double bond, the substituent may be E or Z configuration. If the compound contains a disubstituted cycloalkyl, a cycloalkyl substituent may have a cis- or trans-configuration relative to another substituent of the same cycloalkyl frame.

Any resulting mixtures of stereoisomers can be separated on the basis of the physicochemical differences of the constituents, into the pure or substantially pure geometric isomers, enantiomers, diastereomers, for example, by chromatography and/or fractional crystallization. Any resulting racemates of final products or intermediates can be resolved into the optical antipodes by methods known to those skilled in the art, e.g., by separation of the diastereomeric salts thereof. Racemic products can also be resolved by chiral chromatography, e.g., high performance liquid chromatography (HPLC) using a chiral adsorbent. Preferred enantiomers can also be prepared by asymmetric syntheses. See, for example, Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Principles of Asymmetric Synthesis (2nd Ed. Robert E. Gawley, Jeffrey Aube, Elsevier, Oxford, U K, 2012); Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, N Y, 1962); Wilen, S. H. Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind. 1972); Chiral Separation Techniques: A Practical Approach (Subramanian, G. Ed., Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2007).

Diastereomeric mixtures may be separated into their individual diastereomers on the basis of their physical chemical differences by methods well known to those skilled in the art, such as, for example, by chromatography and/or fractional crystallization. Enantiomers may be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., chiral auxiliary such as a chiral alcohol or Mosher's acid chloride, or formation of a mixture of diastereomeric salts), separating the diastereomers and converting (e.g., hydrolyzing or de-salting) the individual diastereomers to the corresponding pure enantiomers. Enantiomers may also be separated by use of chiral HPLC column.

The compounds of the invention can form pharmaceutically acceptable salts, which are also within the scope of this invention. A “pharmaceutically acceptable salt” refers to a salt of a free acid or base of a compound of Formula (I)a or (I)b that is non-toxic, is physiologically tolerable, is compatible with the pharmaceutical composition in which it is formulated, and is otherwise suitable for formulation and/or administration to a subject. Reference to a compound herein is understood to include reference to a pharmaceutically acceptable salt of said compound unless otherwise indicated.

Compound salts include acidic salts formed with inorganic and/or organic acids, as well as basic salts formed with inorganic and/or organic bases. In addition, where a given compound contains both a basic moiety, such as, but not limited to, a pyridine or imidazole, and an acidic moiety, such as, but not limited to, a carboxylic acid, one of skill in the art will recognize that the compound may exist as a zwitterion (“inner salt”); such salts are included within the term “salt” as used herein. Salts of the compounds of the invention may be prepared, for example, by reacting a compound with an amount of a suitable acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.

Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate (“mesylate”), ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1′-methylene-bis(2-hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counterions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counter ion.

Exemplary acid addition salts include acetates, ascorbates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, fumarates, hydrochlorides, hydrobromides, hydroiodides, lactates, maleates, methanesulfonates, naphthalenesulfonates, nitrates, oxalates, phosphates, propionates, salicylates, succinates, sulfates, tartarates, thiocyanates, toluenesulfonates (also known as tosylates,) and the like.

Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as dicyclohexylamines, tert-butyl amines, and salts with amino acids such as arginine, lysine and the like. Basic nitrogen-containing groups may be quarternized with agents such as lower alkyl halides (e.g., methyl, ethyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, and dibutyl sulfates), long chain halides (e.g., decyl, lauryl, and stearyl chlorides, bromides and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides), and others.

Additionally, acids and bases which are generally considered suitable for the formation of pharmaceutically useful salts from pharmaceutical compounds are discussed, for example, by P. Stahl et al, Camille G. (eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use. (2002) Zurich: Wiley-VCH; S. Berge et al, Journal of Pharmaceutical Sciences (1977) 66(1) 1-19; P. Gould, International J. of Pharmaceutics (1986) 33 201-217; Anderson et al, The Practice of Medicinal Chemistry (1996), Academic Press, New York; and in The Orange Book (Food & Drug Administration, MD, available from FDA). These disclosures are incorporated herein by reference thereto.

Additionally, any compound described herein is intended to refer also to any unsolvated form, or a hydrate, solvate, or polymorph of such a compound, and mixtures thereof, even if such forms are not listed explicitly. “Solvate” means a physical association of a compound of the invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances, the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of a crystalline solid. “Solvate” encompasses both solution-phase and isolatable solvates. Suitable solvates include those formed with pharmaceutically acceptable solvents such as water, ethanol, and the like. In some embodiments, the solvent is water and the solvates are hydrates.

One or more compounds of the invention may optionally be converted to a solvate. Methods for the preparation of solvates are generally known. Thus, for example, M. Caira et al., J. Pharmaceutical Sci., 93(3), 601-611 (2004), describes the preparation of the solvates of the antifungal fluconazole in ethyl acetate as well as from water. Similar preparations of solvates, hemisolvate, hydrates, and the like are described by E. C. van Tonder et al, AAPS PharmSciTech., 5(1), article 12 (2004); and A. L. Bingham et al, Chem. Commun., 603-604 (2001). A typical, non-limiting process involves dissolving the compound of the invention in a suitable amount of the solvent (organic solvent or water or a mixture thereof) at a higher than ambient temperature, and cooling the solution at a rate sufficient to form crystals which are then isolated by standard methods. Analytical techniques such as, for example, infrared spectroscopy, show the presence of the solvent (or water) in the crystals as a solvate (or hydrate).

The present invention also relates to pharmaceutically active metabolites of compounds of Formula (I)a or (I)b, and uses of such metabolites in the methods of the invention. A “pharmaceutically active metabolite” means a pharmacologically active product of metabolism in the body of a compound of Formula (I)a or (I)b or salt thereof. Active metabolites of a compound may be determined using routine techniques known or available in the art. See, e.g., Bertolini et al., J. Med. Chem. 1997, 40, 2011-2016; Shan et al., J. Pharm. Sci. 1997, 86 (7), 765-767; Bagshawe, Drug Dev. Res. 1995, 34, 220-230; Bodor, Adv. Drug Res. 1984, 13, 255-331; Bundgaard, Design of Prodrugs (Elsevier Press, 1985); and Larsen, Design and Application of Prodrugs, Drug Design and Development (Krogsgaard-Larsen et al., eds., Harwood Academic Publishers, 1991).

Any formula given herein is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds. Isotopically labeled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, ³⁶Cl, and ¹²⁵I, respectively. Such isotopically labelled compounds are useful in metabolic studies (for example with ¹⁴C), reaction kinetic studies (with, for example ²H or ³H), detection or imaging techniques [such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients. In particular, an ¹⁸F or ¹¹C labeled compound may be particularly suitable for PET or SPECT studies. Further, substitution with heavier isotopes such as deuterium (i.e., ²H) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements. Isotopically labeled compounds of this invention can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

The use of the terms “salt”, “solvate”, “polymorph”, and the like, with respect to the compounds described herein is intended to apply equally to the salt, solvate, and polymorph forms of enantiomers, stereoisomers, rotamers, tautomers, atropisomers, and racemates of the compounds of the invention.

Abbreviation:

EA Ethyl acetate

DCM Dichloromethane

THF Tetrahydrofuran

TBAF Tetrabutylammonium Fluoride

PE Petroleum Ether

i-PrOH Isopropanol

PivCl Pivaloyl chloride

TEA Triethylamine

DIEA N-Ethyldiisopropylamine

n-BuLi n-Butyllithium

(Boc)₂O Di-tert-butyl dicarbonate

HMDS Hexamethyldisilazide

BnBr Benzyl bromide

HATU O-(7-azabenzotriazol-1-yl)uronium hexafluoro-phosphate

4-AT (2,2,6,6-Tetramethyl-1-oxy-4-piperidinyl)amine

MTBE Methyl tert-butyl ether

KHMDS Potassium bis(trimethylsilyl)amide

TFA Trifluoracetic acid

T₃P 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide

DIADD iisopropyl azodicarboxylate

m-CPBA m-Chloroperbenzoic acid

CbzCl Benzyl Chloroformate

Cbz Carbobenzyl

Fmoc 9-Fluorenylmethoxycarbonyl

Boc tert-butyloxy carbonyl

Bn Benzyl

DMFN N′-dimethylformamide

DMAP 4-(dimethylamino)pyridine

DMSO Dimethyl sulphoxide

DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene

CAN ammonium ceric nitrate

HPLC High performance liquid chromatography

J coupling constant (in NMR)

Min minute(s)

H hour(s)

NMR nuclear magnetic resonance

Prep preparative

t-Bu tert-butyl

iPr isopropyl

TLC thin-layer chromatography

Ala Alanine

Leu Arginine

Ile Isoleucine

Phe Phenylalanine

Met Methionine

Pro Proline

Gly Glycine

Ser Serine

Thr Threonine

Cys Cysteine

Tyr Tyrosine

Asn Asparagine

Gln Glutamine

His Histidine

Lys Lysine

Arg Arginine

Asp Aspartic acid

Glu Glutamic acid

DESCRIPTION OF COMPOUNDS OF THE INVENTION

The present invention relates to particular molecules and pharmaceutically acceptable salts or isomers thereof. The invention further relates to molecules which are useful in modulating dysfunctional glutamate transmission and pharmaceutically acceptable salts, solvates, esters, or isomers thereof.

The invention is directed to compounds as described herein and pharmaceutically acceptable salts, solvates, esters, or isomers thereof, and pharmaceutical compositions comprising one or more compounds as described herein and pharmaceutically acceptable salts or isomers thereof. One aspect of this invention is the provision of compounds, compositions, kits, and antidotes for modulating glutamate transmission in mammals having a compound of Formula (I)a or (I)b, pharmaceutically acceptable salts thereof, and individual enantiomers and diastereomers thereof.

Wherein z is 0 or 1;

L is absent, or selected from a group consisting of: C1-C10 alkyl, C1-C10 alkoxyl, cycloalkyl, C1-C10 alkylcycloalkyl, heterocycloalkyl, C1-C10 alkylheterocycloalkyl, aryl, C1-C10 alkylaryl, heteroaryl, and C1-C10 alkylheteroaryl, wherein said cycloalkyl, or heterocycloalkyl has about 3 to about 7 ring carbons, and said aryl or heteroaryl has about 5 to about 10 ring carbons;

Q is selected from a group consisting of:

wherein n is an integer selected from 0 to 10;

-   -   R₁ or R₂ is H or CH₃;     -   R₃ or R₄ is H or C1-C5alkyl;     -   W is absent, 0 or S; and     -   i) when W is absent, R₃ and R₄ can, optionally, together form         the following structure:

-   -   ii) when W is O, OR₃ and OR₄ can, optionally, together form the         following structure:

PEP is a moiety having the following peptidyl structure:

wherein a * mark denotes a chiral center that can be either in an (S) or (R) configuration and m is an integer selected from 0 to 10;

X₁ is selected from a group consisting of H, —(C═O)—O-Rm, —(SO)—O-Rm, —(SO₂)—O-Rm, —(SO₂)—N-(Rm)₂, and —(C═O)—N-(Rm)₂, where Rm is C₁-C₆ alkyl, C₁-C₆ alkoxyl, C₁-C₆ alkenyl, C₁-C₆ haloalkyl, cycloalkyl, C₁-C₆ alkylcycloalkyl, heterocycloalkyl, C₁-C₆ alkylheterocycloalkyl, aryl, C₁-C₆ alkylaryl, heteroaryl, and C₁-C₆ alkylheteroaryl, wherein said cycloalkyl, or heterocycloalkyl has about 3 to about 7 ring carbons, and said aryl or heteroaryl has about 5 to about 10 ring carbons;

X₂, X₃ or X₄ is selected from a group consisting of C1-C10 alkyl, C1-C10 alkenyl, aryl, C₁-C₆ alkylaryl, heteroaryl, C₁-C₆ alkylheteroaryl, wherein said aryl or heteroaryl having from 5 to 6 ring carbons;

A is absent, or selected from a group consisting of: Ala, Leu, Ile, Phe, Met, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Gln, His, Lys, Arg, Asp, Glu, and Val, each of which can be in either a D or L configuration; wherein the amino residue is either unprotected, or protected by a protecting group selected from a group consisting of Cbz, and Fmoc;

G is absent, or selected from a group consisting of: —O—, —S—, —NH—; —NH—(C═O)—O—, —O—(C═O)—NH—, —NH—(C═O)—NH—, —NHSO₂—, —SO—, and —SO₂—.

In some embodiments, in which compounds have the general Formula (I)a or (I)b, wherein L is a straight or branched chain alkyl having about 1 to about 10 carbon atoms.

In some embodiments, in which compounds have the general Formula (I)a or (I)b, wherein L is a substituted or unsubstituted mono- or bicyclic aryl having about 5 to about 10 ring carbon atoms, or a C1-C10 alkylaryl having about 5 to about 10 ring carbon atoms.

In some embodiments, in which compounds have the general Formula (I)a or (I)b, wherein L is a substituted or unsubstituted mono- or bicyclic heteroaryl having about 5 to about 10 carbon or hetero ring atoms, or a C₁-C₁₀ alkylaryl having about 5 to about 10 ring carbon or hetero ring atoms, wherein hetero atoms include O, N and S; and preferably, L is a substituted or unsubstituted pyridine.

In other embodiments, there is the provision of compounds having the general Formula (I)a or (I)b, wherein G is absent, or is, preferably, —NHC═O—, —NH(C═O)—O— or —NHSO₂—

In still other embodiments, there is the provision of compounds having the general Formula (I)a or (I)b, wherein W is absent, O or S;

In yet still other embodiments, in which compounds have the general Formula (I)a or (I)b, wherein PEP is preferably

wherein a * mark denotes a chiral center that can be either in an (S) or (R) configuration;

X₁ is hydrogen, or selected from the group consisting of —(C═O)—O-Rm, —(SO)—O-Rm, —(SO₂)—O-Rm, —(SO₂)—N-(Rm)₂, and —(C═O)—N-(Rm)₂, where Rm is C₁-C₆ alkyl, C₁-C₆ alkoxyl, C₁-C₆ alkenyl, C₁-C₆ haloalkyl, cycloalkyl, C₁-C₆ alkylcycloalkyl, heterocycloalkyl, C₁-C₆ alkylheterocycloalkyl, aryl, C₁-C₆ alkylaryl, heteroaryl, and C₁-C₆ alkylheteroaryl, wherein said cycloalkyl, or heterocycloalkyl has about 3 to about 7 ring carbons, and said aryl or heteroaryl has about 5 to about 10 ring carbons;

X₂, X₃ or X₄ is selected from a group consisting of C1-C10 alkyl, C₁-C₁₀ alkenyl, aryl, C₁-C₆ alkylaryl, heteroaryl, C₁-C₆ alkylheteroaryl, wherein said aryl or heteroaryl having from 5 to 6 ring carbons;

A is absent, or selected from a group consisting of: Ala, Leu, Ile, Phe, Met, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Gln, His, Lys, Arg, Asp, Glu, and Val, each of which can be in either a D or L configuration; wherein the amino residue is either unprotected, or protected by a protecting group selected from a group consisting of Cbz and Fmoc.

Another embodiment of the invention is the provision of a compound, wherein the various moieties are independently selected, A is, preferably, absent or is Valine.

Yet another embodiment of the invention is the provision of a compound, wherein the various moieties are independently selected, X₁ is, preferably, absent, or selected from a group consisting of: Et-O—(C═O)—, i-Pr-(C═O)—, t-Bu-O—(C═O)— or cyclopropyl-CH₂—O—(C═O)—.

Yet another embodiment of the invention is the provision of a compound, wherein the various moieties are independently selected, X₂ is, preferably, selected from a group consisting of: i-Propyl-CH₂—, cyclopropyl-CH₂—, cyclopentyl-CH₂— or Admantyl-CH₂—.

Yet another embodiment of the invention is the provision of a compound, wherein the various moieties are independently selected, X₃ is, preferably, selected from a group consisting of: Ph-CH₂— or Pyr-CH₂—.

Yet another embodiment of the invention is the provision of a compound, wherein the various moieties are independently selected, X₄ is, preferably, selected from a group consisting of: i-Propyl-CH₂—, cyclopropyl-CH₂— or cyclopentyl-CH₂—.

Yet another embodiment of the invention is the provision of a compound, wherein the various moieties are independently selected, A is absent, X₁ is t-Bu-O—(C═O)—, X₂ is i-Propyl-CH₂—, X₃ is Ph-CH₂—, X₄ is i-Propyl-CH₂—.

Yet another embodiment of the invention is the provision of a compound, wherein the various moieties are independently selected, A is absent, X₁ is cyclopropyl-CH₂—O—(C═O)—, X₂ is i-Propyl-CH₂—, X₃ is Ph-CH₂—, X₄ is i-Propyl-CH₂—.

Yet another embodiment of the invention is the provision of a compound, wherein the various moieties are independently selected, A is absent, X₁ is ethyl-CH₂—O—(C═O)—, X₂ is i-Propyl-CH₂—, X₃ is Ph-CH₂—, X₄ is i-Propyl-CH₂—.

Yet another embodiment of the invention is the provision of a compound, wherein the various moieties are independently selected, A is absent, X₁ is t-Bu-O—(C═O)—, X₂ is i-Propyl-CH₂—, X₃ is Ph-CH₂—, X₄ is cyclopropyl-CH₂—.

Yet another embodiment of the invention is the provision of a compound, wherein the various moieties are independently selected, A is absent, X₁ is t-Bu-O—(C═O)—, X₂ is i-Propyl-CH₂—, X₃ is Ph-CH₂—, X₄ is cyclopentyl-CH₂—.

Yet another embodiment of the invention is the provision of a compound, wherein the various moieties are independently selected, A is absent, X₁ is t-Bu-O—(C═O)—, X₂ is Admantyl-CH₂—; X₃ is Ph-CH₂—, X₄ is i-Propyl-CH₂—.

Yet another embodiment of the invention is the provision of a compound, wherein the various moieties are independently selected, A is valine, X₁ is t-Bu-O—(C═O)—, X2 is i-Propyl-CH₂—, X3 is Ph-CH₂—, X₄ is i-Propyl-CH₂—.

Yet another embodiment of the invention is the provision of a compound, wherein the various moieties are independently selected, A is valine, X₁ is cyclopropyl-CH₂—O—(C═O)—, X₂ is i-Propyl-CH₂—, X₃ is Ph-CH₂—, X₄ is i-Propyl-CH₂—.

Yet another embodiment of the invention is the provision of a compound, wherein the various moieties are independently selected, A is valine, X₁ is cyclopropyl-CH₂—O—(C═O)—, X₂ is i-Propyl-CH₂—, X₃ is Ph-CH₂—, X₄ is cyclopropyl-CH₂—.

Yet another embodiment of the invention is the provision of a compound, wherein the various moieties are independently selected, A is valine, X₁ is cyclopropyl-CH₂—O—(C═O)—, X₂ is i-Propyl-CH₂—, X₃ is Ph-CH₂—, X₄ is cyclopentyl-CH₂—.

Yet another embodiment of the invention is the provision of a compound, wherein the various moieties are independently selected, A is valine, X₁ is t-Bu-O—(C═O)—, X₂ is Admantyl-CH₂—; X₃ is Ph-CH₂—, X₄ is i-Propyl-CH₂—.

In certain embodiments, the compound of Formula (I)a or (I)b is further illustrated by the following compound group consisting of:

pharmaceutically acceptable salts thereof.

An aspect of the present invention concerns compounds disclosed herein.

An aspect of the present invention concerns compounds which are or can be modulators of ferroptosis.

An aspect of the present invention concerns the use of compounds disclosed herein for the preparation of a medicament used in the treatment, prevention, inhibition or elimination of tumors.

An aspect of the present invention concerns the use of compounds disclosed herein as a modulator of ferroptosis for the preparation of a medicament used in the treatment, prevention, inhibition or elimination of a disorder or disease or medical condition in a patient by modulating ferroptosis in said patient.

The present invention also describes one or more methods of synthesizing the compounds of the present invention.

The invention also describes one or more uses of the compounds of the present invention.

The invention also describes one or more uses of the compounds of the present invention with an adjunctive agent.

The present invention also describes one or more methods of preparing various pharmaceutical compositions comprising the compounds of the present invention.

The invention also describes one or more uses of the various pharmaceutical compositions of the present invention for the preparation of a medicament used in the treatment, prevention, inhibition or elimination of a disorder or disease or medical condition in a patient by modulating ferroptosis in said Patient.

Pharmaceutical Composition of the Compound of the Invention and Preparations and Administration

The present invention provides a pharmaceutical composition comprising compounds of the present invention, e.g., example compounds. According to the specific examples of the present invention, the pharmaceutical composition can further comprise pharmaceutically acceptable excipient, carrier, adjuvant, solvent and a combination thereof.

The present invention provides a method of treating, preventing or ameliorating a disease or disorder, comprising administrating a safe and effective amount of a pharmaceutical composition containing compounds of the invention with, optionally, one or more adjunctive therapeutic active agents. The amount of the compound of the pharmaceutical composition disclosed herein refers to an amount which can be effectively detected to modulate ferroptosis in biological samples and in a patient. The active ingredient may be administered to subjects in need of such treatment in dosage that will provide optimal pharmaceutical efficacy, which is not limited to the desired therapeutic effects, on the route of administration, and on the duration of the treatment. The dosage will vary from patient to patient depending upon the nature and severity of disease, the patient's weight, special diet then being followed by a patient, concurrent medication, and other factors which those skilled in the art will recognize.

The actual dosage employed may be varied depending upon the requirements of the patient and the severity of the condition being treated. Determination of the proper dosage regimen for a particular situation is within the skill of the art. For convenience, the total daily dosage may be divided and administered in portions during the day as required. The amount and frequency of administration of the compounds of the invention and/or the pharmaceutically acceptable salts thereof will be regulated according to the judgment of the attending clinician considering such factors as age, condition and size of the patient as well as severity of the symptoms being treated.

It will also be appreciated that certain of the compounds of the present invention can exist in free form for treatment, or where appropriate, as a pharmaceutically acceptable derivative or a prodrug thereof. A pharmaceutically acceptable derivative includes pharmaceutically acceptable salts, esters, salts of such esters, or any other adduct or derivative which upon administration to a patient in need thereof provide, directly or indirectly, a compound as otherwise described herein, or a therapeutically effective metabolite or residue thereof.

The pharmaceutical compositions of the invention may be prepared and packaged in bulk form wherein a safe and effective amount of a compound of Formula (I)a or (I)b disclosed herein can be extracted and then given to the patient, such as with powders or syrups. Generally, dosage levels of between 0.0001 to 10 mg/kg of body weight daily are administered to the patient to obtain effective modulation of dysfunctional glutamate transmission. Alternatively, the pharmaceutical compositions of the invention may be prepared and packaged in unit dosage form wherein each physically discrete unit contains a safe and effective amount of a compound of Formula (I)a or (I)b disclosed herein.

When the pharmaceutical compositions of the present invention also contain one or more other active ingredients, in addition to a compound of the present invention, the weight ratio of the compound of the present invention to the second active ingredient may be varied and depend upon the effective dose of each ingredient. Thus, for example, when a compound of the present invention is combined with another agent, the weight ratio of the compound of the present invention to the other agent will generally range from about 1000:1 to about 1:1000, such as about 200:1 to 1:200. Combinations of a compound of the present invention and other active ingredients will generally also be within the aforementioned range, but in each case, an effective dose of each active ingredient in the combination should be used.

“Pharmaceutically acceptable excipient” as used herein means a pharmaceutically acceptable material, composition or vehicle involved in giving form or consistency to the pharmaceutical composition. Each excipient must be compatible with the other ingredients of the pharmaceutical composition when commingled, such that interactions which would substantially reduce the efficacy of the compound of the invention when administered to a patient and would result in pharmaceutically unacceptable compositions. In addition, each excipient must of course be of sufficiently high purity to render it pharmaceutically acceptable.

Suitable pharmaceutically acceptable excipients will vary depending upon the particular dosage form chosen. In addition, suitable pharmaceutically acceptable excipients may be chosen for a particular function that they may serve in the composition. For example, certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of uniform dosage forms. Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of stable dosage forms. Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the carrying or transporting the compound of the present invention once administered to the patient from one organ, or portion of the body, to another organ, or portion of the body. Certain pharmaceutically acceptable excipients may be chosen for their ability to enhance patient compliance.

Suitable pharmaceutically acceptable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, flavor masking agents, coloring agents, anticaking agents, humectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents. The skilled artisan will appreciate that certain pharmaceutically acceptable excipients may serve more than one function and may serve alternative functions depending on how much of the excipient is present in the formulation and what other ingredients are present in the formulation.

Skilled artisans possess the knowledge and skill in the art to enable them to select suitable pharmaceutically acceptable excipients in appropriate amounts for use in the invention. In addition, there are resources that are available to the skilled artisan that describe pharmaceutically acceptable excipients and may be useful in selecting suitable pharmaceutically acceptable excipients. Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company), The Handbook of Pharmaceutical Additives (Gower Publishing Limited), and The Handbook of Pharmaceutical Excipients (the American Pharmaceutical Association and the Pharmaceutical Press).

In Remington: The Science and Practice of Pharmacy, 21st edition, 2005, ed. D. B. Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York, the contents of each of which is incorporated by reference herein, are disclosed various carriers used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this invention.

The pharmaceutical compositions of the invention are prepared using techniques and methods known to those skilled in the art. Some of the methods commonly used in the art are described in Remington's Pharmaceutical Sciences (Mack Publishing Company).

Therefore, another aspect of the present invention is related to a method for preparing a pharmaceutical composition. The pharmaceutical composition contains the compound disclosed herein and pharmaceutically acceptable excipient, carrier, adjuvant, vehicle or a combination thereof, the method comprises mixing various ingredients. The pharmaceutical composition containing the compound disclosed herein can be prepared for example at normal ambient temperature and pressure.

The compound of the invention will typically be formulated into a dosage form adapted for administration to the patient by the desired route of administration. For example, dosage forms include those adapted for (1) oral administration such as tablets, capsules, caplets, pills, troches, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and cachets; (2) parenteral administration such as sterile solutions, suspensions, and powders for reconstitution; (3) transdermal administration such as transdermal patches; (4) rectal administration such as suppositories; (5) inhalation such as aerosols, solutions, and dry powders; and (6) topical administration such as creams, ointments, lotions, solutions, pastes, sprays, foams, and gels.

The pharmaceutical compositions provided herein may be provided as compressed tablets, tablet triturates, chewable lozenges, rapidly dissolving tablets, multiple compressed tablets, or enteric-coating tablets, sugar-coated, or film-coated tablets. Enteric-coated tablets are compressed tablets coated with substances that resist the action of stomach acid but dissolve or disintegrate in the intestine, thus protecting the active ingredients from the acidic environment of the stomach. Enteric-coatings include, but are not limited to, fatty acids, fats, phenylsalicylate, waxes, shellac, ammoniated shellac, and cellulose acetate phthalates. Sugar-coated tablets are compressed tablets surrounded by a sugar coating, which may be beneficial in covering up objectionable tastes or odors and in protecting the tablets from oxidation. Film-coated tablets are compressed tablets that are covered with a thin layer or film of a water-soluble material. Film coatings include, but are not limited to, hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000, and cellulose acetate phthalate. Film coating imparts the same general characteristics as sugar coating. Multiple compressed tablets are compressed tablets made by more than one compression cycle, including layered tablets, and press-coated or dry-coated tablets.

The tablet dosage forms may be prepared from the active ingredient in powdered, crystalline, or granular forms, alone or in combination with one or more carriers or excipients described herein, including binders, disintegrants, controlled-release polymers, lubricants, diluents, and/or colorants. Flavoring and sweetening agents are especially useful in the formation of chewable tablets and lozenges.

The pharmaceutical compositions provided herein may be provided as soft or hard capsules, which can be made from gelatin, methylcellulose, starch, or calcium alginate. The hard gelatin capsule, also known as the dry-filled capsule (DFC), consists of two sections, one slipping over the other, thus completely enclosing the active ingredient. The soft elastic capsule (SEC) is a soft, globular shell, such as a gelatin shell, which is plasticized by the addition of glycerin, sorbitol, or a similar polyol. The soft gelatin shells may contain a preservative to prevent the growth of microorganisms. Suitable preservatives are those as described herein, including methyl- and propyl-parabens, and ascorbic acid. The liquid, semisolid, and solid dosage forms provided herein may be encapsulated in a capsule. Suitable liquid and semisolid dosage forms include solutions and suspensions in propylene carbonate, vegetable oils, or triglycerides. Capsules containing such solutions can be prepared as described in U.S. Pat. Nos. 4,328,245; 4,409,239; and 4,410,545. The capsules may also be coated as known by those of skill in the art to modify or sustain dissolution of the active ingredient.

The pharmaceutical compositions provided herein may be provided in liquid and semisolid dosage forms, including emulsions, solutions, suspensions, elixirs, and syrups. An emulsion is a two-phase system, in which one liquid is dispersed in the form of small globules throughout another liquid, which can be oil-in-water or water-in-oil. Emulsions may include a pharmaceutically acceptable non-aqueous liquids or solvent, emulsifying agent, and preservative. Suspensions may include a pharmaceutically acceptable suspending agent and preservative. Aqueous alcoholic solutions may include a pharmaceutically acceptable acetal, such as a di(lower alkyl)acetal of a lower alkyl aldehyde, e.g., acetaldehyde diethyl acetal; and a water-miscible solvent having one or more hydroxy groups, such as propylene glycol and ethanol. Elixirs are clear, sweetened, and hydroalcoholic solutions. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may also contain a preservative. For a liquid dosage form, for example, a solution in a polyethylene glycol may be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g., water, to be measured conveniently for administration.

Other useful liquid and semisolid dosage forms include, but are not limited to, those containing the active ingredient(s) provided herein, and a dialkylated mono- or poly-alkylene glycol, including, 1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether, wherein 350, 550, and 750 refer to the approximate average molecular weight of the polyethylene glycol. These formulations may further comprise one or more antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, bisulfite, sodium metabisulfite, thiodipropionic acid and its esters, and dithiocarbamates.

Where appropriate, dosage unit formulations for oral administration can be microencapsulated. The formulation can also be prepared to prolong or sustain the release as for example by coating or embedding particulate material in polymers, wax, or the like.

The pharmaceutical compositions provided herein for oral administration may be also provided in the forms of liposomes, micelles, microspheres, or nanosystems. Micellar dosage forms can be prepared as described in U.S. Pat. No. 6,350,458.

The pharmaceutical compositions provided herein may be provided as non-effervescent or effervescent, granules and powders, to be reconstituted into a liquid dosage form. Pharmaceutically acceptable carriers and excipients used in the non-effervescent granules or powders may include diluents, sweeteners, and wetting agents. Pharmaceutically acceptable carriers and excipients used in the effervescent granules or powders may include organic acids and a source of carbon dioxide.

Coloring and flavoring agents can be used in all above dosage forms.

The compounds disclosed herein can also be coupled to soluble polymers as targeted medicament carriers. Such polymers may encompass polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidophenol, polyhydroxyethylaspartamidophenol or polyethylene oxide polylysine, substituted by palmitoyl radicals. The compounds may furthermore be coupled to a class of biodegradable polymers which are suitable for achieving controlled release of a medicament, for example polylactic acid, poly-epsilon-caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydroxypyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.

The pharmaceutical compositions provided herein may be formulated as immediate or modified release dosage forms, including delayed-, sustained, pulsed-, controlled, targeted-, and programmed-release forms.

The pharmaceutical compositions provided herein may be co-formulated with other active ingredients which do not impair the desired therapeutic action, or with substances that supplement the desired action.

The pharmaceutical compositions provided herein may be administered parenterally by injection, infusion, or implantation, for local or systemic administration. Parenteral administration, as used herein, include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial, and subcutaneous administration.

The pharmaceutical compositions provided herein may be formulated in any dosage forms that are suitable for parenteral administration, including solutions, suspensions, emulsions, micelles, liposomes, microspheres, nanosystems, and solid forms suitable for solutions or suspensions in liquid prior to injection. Such dosage forms can be prepared according to conventional methods known to those skilled in the art of pharmaceutical science (see, Remington: The Science and Practice of Pharmacy, supra).

The pharmaceutical compositions intended for parenteral administration may include one or more pharmaceutically acceptable carriers and excipients, including, but not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against the growth of microorganisms, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, cryoprotectants, lyoprotectants, thickening agents, pH adjusting agents, and inert gases.

Suitable aqueous vehicles include, but are not limited to, water, saline, physiological saline or phosphate buffered saline (PBS), sodium chloride injection, Ringers injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringers injection. Non-aqueous vehicles include, but are not limited to, fixed oils of vegetable origin, castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, and medium-chain triglycerides of coconut oil, and palm seed oil. Water-miscible vehicles include, but are not limited to, ethanol, 1,3-butanediol, liquid polyethylene glycol (e.g., polyethylene glycol 300 and polyethylene glycol 400), propylene glycol, glycerin, N-methyl-2-pyrrolidone, N, N-dimethylacetamide, and dimethyl sulfoxide.

Suitable antimicrobial agents or preservatives include, but are not limited to, phenols, cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride (e.g., benzethonium chloride), methyl- and propyl-parabens, and sorbic acid. Suitable isotonic agents include, but are not limited to, sodium chloride, glycerin, and dextrose. Suitable buffering agents include, but are not limited to, phosphate and citrate. Suitable antioxidants are those as described herein, including bisulfite and sodium metabisulfite. Suitable local anesthetics include, but are not limited to, procaine hydrochloride. Suitable suspending and dispersing agents are those as described herein, including sodium carboxymethylcelluose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Suitable emulsifying agents include those described herein, including polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate 80 and triethanolamine oleate. Suitable sequestering or chelating agents include, but are not limited to EDTA. Suitable pH adjusting agents include, but are not limited to, sodium hydroxide, hydrochloric acid, citric acid, and lactic acid. Suitable complexing agents include, but are not limited to, cyclodextrins, including α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, and sulfobutylether 7-β-cyclodextrin (CAPTISOL®, CyDex, Lenexa, Kans.).

The pharmaceutical compositions provided herein may be formulated for single or multiple dosage administration. The single dosage formulations are packaged in an ampoule, a vial, or a syringe. The multiple dosage parenteral formulations must contain an antimicrobial agent at bacteriostatic or fungistatic concentrations. All parenteral formulations must be sterile, as known and practiced in the art.

In one embodiment, the pharmaceutical compositions are provided as ready-to-use sterile solutions. In another embodiment, the pharmaceutical compositions are provided as sterile dry soluble products, including lyophilized powders and hypodermic tablets, to be reconstituted with a sterile vehicle prior to use. In yet another embodiment, the pharmaceutical compositions are provided as ready-to-use sterile suspensions. In yet another embodiment, the pharmaceutical compositions are provided as sterile dry insoluble products to be reconstituted with a vehicle prior to use. In still another embodiment, the pharmaceutical compositions are provided as ready-to-use sterile emulsions.

The pharmaceutical compositions may be formulated as a suspension, solid, semi-solid, or thixotropic liquid, for administration as an implanted depot. In one embodiment, the pharmaceutical compositions provided herein are dispersed in a solid inner matrix, which is surrounded by an outer polymeric membrane that is insoluble in body fluids but allows the active ingredient in the pharmaceutical compositions diffuse through.

Suitable inner matrixes include polymethylmethacrylate, polybutyl-methacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers, such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinyl alcohol, and cross-linked partially hydrolyzed polyvinyl acetate.

Suitable outer polymeric membranes include polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride, vinyl chloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer.

In other aspect, the pharmaceutical composition of the invention is prepared to a dosage form adapted for administration to a patient by inhalation, for example as a dry powder, an aerosol, a suspension, or a solution composition. In one embodiment, the invention is directed to a dosage form adapted for administration to a patient by inhalation as a dry powder. In one embodiment, the invention is directed to a dosage form adapted for administration to a patient by inhalation as a dry powder. Dry powder compositions for delivery to the lung by inhalation typically comprise a compound disclosed herein or a pharmaceutically acceptable salt thereof as a finely divided powder together with one or more pharmaceutically-acceptable excipients as finely divided powders. Pharmaceutically-acceptable excipients particularly suited for use in dry powders are known to those skilled in the art and include lactose, starch, mannitol, and mono-, di-, and polysaccharides. The finely divided powder may be prepared by, for example, micronisation and milling Generally, the size-reduced (e.g. micronised) compound can be defined by a D₅₀ value of about 1 to about 10 microns (for example as measured using laser diffraction).

Aerosols may be formed by suspending or dissolving a compound disclosed herein or a pharmaceutically acceptable salt thereof in a liquified propellant. Suitable propellants include halocarbons, hydrocarbons, and other liquefied gases. Representative propellants include: trichlorofluoromethane (propellant 11), dichlorofluoromethane (propellant 12), dichlorotetrafluoroethane (propellant 114), tetrafluoroethane (HFA-134a), 1,1-difluoroethane (HFA-152a), difluoromethane (HFA-32), pentafluoroethane (HFA-12), heptafluoropropane (HFA-227a), perfluoropropane, perfluorobutane, perfluoropentane, butane, isobutane, and pentane. Aerosols comprising a compound of Formula (I)a or (I)b or a pharmaceutically acceptable salt thereof will typically be administered to a patient via a metered dose inhaler (MDI). Such devices are known to those skilled in the art.

The aerosol may contain additional pharmaceutically-acceptable excipients typically used with MDIs such as surfactants, lubricants, cosolvents and other excipients to improve the physical stability of the formulation, to improve valve performance, to improve solubility, or to improve taste.

Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the patient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6), 318 (1986).

Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. Ointments, creams and gels, may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agent and/or solvents. Such bases may thus, for example, include water and/or an oil such as liquid paraffin or a vegetable oil such as arachis oil or castor oil, or a solvent such as polyethylene glycol. Thickening agents and gelling agents which may be used according to the nature of the base include soft paraffin, aluminum stearate, cetostearyl alcohol, polyethylene glycols, woolfat, beeswax, carboxypolymethylene and cellulose derivatives, and/or glyceryl monostearate and/or non-ionic emulsifying agents.

Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents or thickening agents.

Powders for external application may be formed with the aid of any suitable powder base, for example, talc, lactose or starch. Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilising agents, suspending agents or preservatives.

Topical preparations may be administered via one or more applications per day to the affected area; over skin areas occlusive dressings may advantageously be used. Continuous or prolonged delivery may be achieved via an adhesive reservoir system.

Uses of the Compounds and Compositions of the Invention

Compounds or pharmaceutical compositions of the invention disclosed herein can be used in an amount which can be effectively detected to modulate ferroptosis in biological samples, or a related disorder or disease in a subject. Such uses may include in the manufacture of a medicament for treating, preventing, ameliorating or mitigating a disorder or disease in a subject, as well as other medicaments for modulating ferroptosis, and the compounds of this invention have superior pharmacokinetic and pharmacodynamic properties, fewer toxic side-effect.

Specifically, the amount of the compound of compositions of the present invention may be shown to effectively and detectably modulate ferroptosis in biological samples, or a related disorder or disease in a subject. The compounds or pharmaceutical compositions of the invention may be used for preventing, treating or alleviating diseases relating to ferroptosis in patients.

In one embodiment, the therapies disclosed herein comprise administrating a safe and effective amount of the compound of the invention or the pharmaceutical composition containing the compound of the invention to patients in need. Each example disclosed herein comprises the method of treating the diseases above comprising administrating a safe and effective amount of the compound of the invention or the pharmaceutical composition containing the compound of the invention to patients in need.

In one embodiment, the compound of the invention or the pharmaceutical composition thereof may be administered by any suitable route of administration, including both systemic administration and topical administration. Systemic administration includes oral administration, parenteral administration, transdermal administration and rectal administration. Parenteral administration refers to routes of administration other than enteral or transdermal, and is typically by injection or infusion. Parenteral administration includes intravenous, intramuscular, and subcutaneous injection or infusion. Topical administration includes application to the skin as well as intraocular, intravaginal, inhaled and intranasal administration. In one embodiment, the compound of the invention or the pharmaceutical composition thereof may be administered orally. In another embodiment, the compound of the invention or the pharmaceutical composition thereof may be administered by inhalation. In a further embodiment, the compound of the invention or the pharmaceutical composition thereof may be administered intranasal.

In one embodiment, the compound of the invention or the pharmaceutical composition thereof may be administered once or according to a dosing regimen wherein multiple doses are administered at varying intervals of time for a given period of time. For example, doses may be administered one, two, three, or four times per day. In one embodiment, a dose is administered once per day. In a further embodiment, a dose is administered twice per day. Doses may be administered until the desired therapeutic effect is achieved or indefinitely to maintain the desired therapeutic effect. Suitable dosing regimens for the compound of the invention or the pharmaceutical composition thereof depend on the pharmacokinetic properties of that compound, such as its absorption, distribution, and half-lives of metabolism and elimination, which can be determined by the skilled artisan. In addition, suitable dosing regimens, including the duration such regimens are administered, for the compound of the invention or the pharmaceutical composition thereof depend on the disorder being treated, the severity of the disorder being treated, the age and physical condition of the patient being treated, the medical history of the patient to be treated, the nature of concurrent therapy, the desired therapeutic effect, and like factors within the knowledge and expertise of the skilled artisan. It will be further understood by such skilled artisans that suitable dosing regimens may require adjustment given an individual patient's tolerance to the dosing regimen or over time as individual patient needs change.

The compounds of the present invention may be administered either simultaneously with, or before or after, one or more other therapeutic agents. The compounds of the present invention may be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition as the other agents.

The pharmaceutical composition or combination of the present invention can be in unit dosage of about 1-1000 mg of active ingredients for a subject of about 50-70 kg, preferably about 1-500 mg or about 1-250 mg or about 1-150 mg or about 0.5-100 mg or about 1-50 mg of active ingredients. The therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease.

The above-cited dosage properties can be correlated with in vitro and in vivo tests using advantageously mammals, e.g., mice, rats, dogs, non-human primates, such as monkeys or isolated organs, tissues and preparations thereof. The compounds of the present invention can be applied in vitro in the form of solutions, e.g., preferably aqueous solutions, and in vivo via topically, inhalingly, enterally or parenterally, advantageously intravenously, e.g., as a suspension or in aqueous solution.

In one embodiment, a therapeutically effective dosage of the compound disclosed herein from about 0.1 mg to about 1,000 mg per day. The pharmaceutical compositions should provide a dosage of from about 0.1 mg to about 1,000 mg of the compound. In a special embodiment, pharmaceutical dosage unit forms are prepared to provide from about 1 mg to about 1,000 mg, about 10 mg to about 500 mg, about 20 mg to about 200 mg, about 25 mg to about 100 mg, or about 30 mg to about 60 mg of the active ingredient or a combination of essential ingredients per dosage unit form. In a special embodiment, pharmaceutical dosage unit forms are prepared to provide about 1 mg, 5 mg, 10 mg, 20 mg, 25 mg, 50 mg, 100 mg, 250 mg, 500 mg, 1000 mg of the active ingredient.

Preferred Embodiment of the Invention General Synthetic Procedures

The following examples are provided so that the invention might be more fully understood. However, it should be understood that these embodiments merely provide a method of practicing the present invention, and the present invention is not limited to these embodiments.

Generally, the compounds disclosed herein may be prepared by methods described herein, wherein the substituents are as defined for Formula (I)a or (I)b above, except where further noted. The following non-limiting schemes and examples are presented to further exemplify the invention.

Professionals skilled in the art will recognize that the chemical reactions described may be readily adapted to prepare a number of other compounds disclosed herein, and alternative methods for preparing the compounds disclosed herein are deemed to be within the scope disclosed herein. Those having skill in the art will recognize that the starting materials may be varied and additional steps employed to produce compounds encompassed by the present inventions, as demonstrated by the following examples. In some cases, protection of certain reactive functionalities may be necessary to achieve some of the above transformations. In general, such need for protecting groups, as well as the conditions necessary to attach and remove such groups, will be apparent to those skilled in the art of organic synthesis. For example, the synthesis of non-exemplified compounds according to the invention may be successfully performed by modifications apparent to those skilled in the art, by appropriately protecting interfering groups, by utilizing other suitable reagents known in the art other than those described, and/or by making routine modifications of reaction conditions. Alternatively, the known reaction conditions or the reaction disclosed in the present invention will be recognized as having applicability for preparing other compounds disclosed here.

In the examples described below, unless otherwise indicated all temperatures are set forth in degrees Celsius. Reagents were purchased from commercial suppliers such as Aldrich Chemical Company, Arco Chemical Company and Alfa Chemical Company, and were used without further purification unless otherwise indicated.

Preparation of compounds

Compounds of the present invention, including salts, esters, hydrates, or solvates thereof, can be prepared using any known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes.

The reactions for preparing compounds of the present invention can be carried out in suitable solvents, which can be readily selected by one skilled in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, e.g., temperatures that can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected by a skilled artisan.

Reactions can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or by chromatographic methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectroscopy (LCMS), or thin layer chromatography (TLC). Compounds can be purified by those skilled in the art by a variety of methods, including high performance liquid chromatography (HPLC) (“Preparative LC-MS Purification: Improved Compound Specific Method Optimization” Karl F. Blom, Brian Glass, Richard Sparks, Andrew P. Combs J. Combi. Chem. 2004, 6(6), 874-883, which is incorporated herein by reference in its entirety) and atmospheric pressure column chromatography using silica gel.

Compounds of the present invention can be synthesized using the methods described below, together with synthetic methods known in the art of synthetic organic chemistry, or variations thereon as appreciated by those skilled in the art. Preferred methods include but are not limited to those methods described below. Specifically, the compounds of the present invention of Formula (I)a or (I)b can be synthesized by following the steps outlined in the exemplary general synthetic schemes listed below, and the abbreviations for the reactants or for the chemical groups of the reactants included in the synthetic schemes are defined in the Examples.

The synthesis towards structure A1 can be conducted according to the relevant procedures disclosed in references (1, Journal of the American Chemical Society, 2005, 127, 12460-12461; 2, Organic Letter, 2011, 13, 2318-2321; Journal of Organic Chemistry, 2010, 75, 941-944), but is not limited to these disclosed procedures. Sulfonamide derivatives 1 condensed with compound 2 at the presence of Cp₂ZrHCl to give the enantiomer compound 3, which is further removed the protecting group and oxidized to acid compound 4. With the help of Evans chiral auxiliary, another chiral center was added onto the compound 4 to furnish structure A1.

The structure A2 is also can be conducted according to the alternative route synthesis according to the relevant procedures disclosed in references (Journal of Organic Chemistry, 2012, 77, 6358-6364; Organic Process Research & Development 2010, 14, 441-458; Journal of Organic Chemistry, 1994, 59, 1139-1148), but is not limited to these disclosed procedures. Compound 5 with the help of Evans chiral auxiliary, a chiral center was added onto the compound 6, which is further oxide to aldehyde 7. Compound 7 was condensed with compound 4 via the mechanism of Julia olefination reaction to give compound 9, which is further oxidized to structure A2.

The synthesis towards structure B can be conducted according to the relevant procedures disclosed in references (Bulletin of the Chemical Society of Japan, 1993, 66, 3113-3115; U.S. Pat. No. 7,528,174B2), but is not limited to these disclosed procedures.

The synthesis towards structure C can be conducted according to the relevant procedures disclosed in references (ACS Central Science, 2016, 2 (9), pp 653-659; Journal of Medicinal Chemistry, 1986, 959-971; Journal of Medicinal Chemistry, 1984, 27, 684-691), but is not limited to these disclosed procedures.

The synthesis towards structure D can be conducted according to the relevant procedures disclosed in references (US2007161573A1; US2009042808A1; Journal of Medicinal Chemistry, 1984, 27, 1351-1354), but is not limited to these disclosed procedures.

The synthesis towards structure E can be conducted according to the relevant procedures disclosed in references (US2007161573A1; US2009042808A1; Journal of the American Chemical Society, 2005, 127, 5742-5743; Journal of Organic Chemistry, 2004, 69, 7851-7859), but is not limited to these disclosed procedures.

The synthesis towards structure F can be conducted according to the relevant procedures disclosed in references (US2007161573A1; Journal of the American Chemical Society, 2005, 127, 5742-5743; International Journal of Peptide and Protein Research, 1996; 47, 460-466), but is not limited to these disclosed procedures.

The synthesis towards structure H can be conducted according to the relevant procedures disclosed in references (US2007161573A1; Journal of Pharmacology and Experimental Therapeutics, 2007, 320, 1050-1060), but is not limited to these disclosed procedures.

Preparation and Characterization of Exemplary Compounds

Compounds encompassed in the present disclosure may be prepared via different schemes. Detailed preparation processes of those exemplary compounds via various schemes are described below and the characterization results are listed as well.

Unless stated otherwise, all reagents were purchased from commercial suppliers without further purification. Solvent drying by standard methods was employed when necessary. The plates used for thin-layer chromatography (TLC) were E. Merck silica gel 60F254 (0.24 nm thickness) precoated on aluminum plates, and then visualized under UV light (365 nm and 254 nm) or through staining with a 5% of dodecamolybdophosphoric acid in ethanol and subsequent heating. Column chromatography was performed using silica gel (200-400 mesh) from commercial suppliers. ¹H NMR spectra were recorded on an Agilent 400-MR NMR spectrometer (400.00 MHz for 1 H) at room temperature. Solvent signal was used as reference for ¹H NMR (CDCl₃, 7.26 ppm; CD₃OD, 3.31 ppm; DMSO-d6, 2.50 ppm; D₂O, 4.79 ppm). The following NMR acronyms and abbreviations were used to explain the multiplicities: s=singlet, d=doublet, t=triplet, q=quartet, br. s.=broad singlet, dd=double doublet, td=triple doublet, dt=double triplet, dq=double quartet, m=multiplet.

EXAMPLES

It should be noted that embodiments of the present invention described in detail below are exemplary for explaining the present invention only, and not be construed as limiting the present scope of invention. Examples without a specific technology or condition can be implemented according to technology or condition in the documentation of the art or according to the product instructions. The reagents or instruments without manufacturers are available through conventional purchase. Those having skill in the art will recognize that the starting materials may be varied and additional steps employed to produce compounds encompassed by the present inventions, as demonstrated by the following examples.

Example 1: tert-Butyl ((4S,7S,E)-7-benzyl-8-((S)-2-(((S)-6-(3-(4,5-dimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propanamido)-1-(isopropylamino)-1-oxohexan-2-yl)carbamoyl)pyrrolidin-1-yl)-2-methyl-8-oxooct-5-en-4-yl)carbamate (I) tert-butyl N-[(4S,5E,7S)-7-benzyl-8-[(2S)-2-{[(1S)-5-({[3-(4,5-dimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propoxy]carbonyl}amino)-1-[propan-2-yl)carbamoyl]pentyl]carbamoyl}pyrrolidin-1-yl]-2-methyl-8-oxooct-5-en-4-yl]carbamate (II) tert-butyl N-[(4S,5E,7S)-7-benzyl-8-[(2S)-2-{[(1S)-5-[({[10-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)decyl]oxy}carbonyl)amino]-1-[(propan-2-yl)carbamoyl]pentyl]carbamoyl}pyrrolidin-1-yl]-2-methyl-8-oxooct-5-en-4-yl]carbamate (III)

Step 1: Synthesis of (S)-(9H-fluoren-9-yl)methyl tert-butyl (6-(isopropylamino)-6-oxohexane-1,5-diyl)dicarbamate (52)

To a solution of compound 51 (500 mg, 1.07 mmol) and propan-2-amine (76 mg, 1.28 mmol) in CH₂Cl₂ (10 mL) at 0° C. was added T₃P (714 mg, 1.28 mmol) and DIEA (165 mg, 1.28 mmol). The reaction mixture was allowed to warm to 25° C. and stirred for 16 h. The reaction mixture was quenched with saturated aqueous NH₄Cl solution (20 mL), and extracted by EtOAc (3×10 mL). The organic layer was washed with aqueous (5%) NaHCO₃ solution, aqueous HCl (10 mL, 1 M), brine solution (3×10 mL), and dried over Na₂SO₄. The residues were concentrated in vacuum to give product 52 (480 mg, 88%) as a white foam. MS (ESI): [M+H⁺]=496.2.

Step 2: Synthesis of (S)-(9H-fluoren-9-yl)methyl (5-amino-6-(isopropylamino)-6-oxohexyl)carbamate (53)

A solution of compound 52 (480 mg, 0.94 mmol) in HCl in MeOH (10 mL, 3 M) was stirred at 25° C. for 2 h and then the solvents were removed in vacuum to give product 53 (384 mg, 100%) as a white foam. MS (ESI): [M+H⁺]=396.1.

Step 3: Synthesis of (S)-tert-butyl 2-(((R)-6-((((9H-fluoren-9-yl)methoxy)carbonyl) amino)-1-(isopropylamino)-1-oxohexan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (54)

To a solution of compound 53 (384 mg, 0.94 mmol) and (S)-1-(tert-butoxycarbonyl) pyrrolidine-2-carboxylic acid (243 mg, 1.1.13 mmol) in CH₂Cl₂ (10 mL) at 0° C. was added T₃P (720 mg, 1.13 mmol) and DIEA (146 mg, 1.13 mmol). The reaction mixture was allowed to warm to 25° C. and stirred for 16 h. The reaction mixture was quenched with saturated aqueous NH₄Cl solution (10 mL), and extracted by EtOAc (3×10 mL). The organic layer was washed with aqueous (5%) NaHCO₃ solution (10 mL), aqueous HCl (2×10 mL, 1 M), saturated aqueous brine solution (3×10 mL), and dried over Na₂SO₄. The organic layer was concentrated in vacuum to give the product 54 (530 mg, 87%) as a white foam. MS (ESI): [M+H⁺]=593.4.

Step 4: Synthesis of (9H-fluoren-9-yl)methyl ((S)-6-(isopropylamino)-6-oxo-5-((S)-pyrrolidine-2-carboxamido)hexyl)carbamate (55)

A solution of compound 54 (250 mg, 0.41 mmol) in HCl in MeOH (10 mL, 3 M) was stirred at 25° C. for 2 h and then concentrated in vacuum to give product 55 (207 mg, 100%) as a white foam. MS (ESI): [M+H⁺]=493.3.

Step 5: Synthesis of (9H-fluoren-9-yl)methyl N-[(5S)-5-{[(2S)-1-[(2S,3E,5S)-2-benzyl-5-{[(tert-butoxy)carbonyl]amino}-7-methyloct-3-enoyl]pyrrolidin-2-yl]formamido}-5-[(pro-pan-2-yl)carbamoyl]pentyl]carbamate (56)

To a solution of compound 55 (207 mg, 0.41 mmol) and (2S,5S,E)-2-benzyl-5-((tert-butoxycarbonyl)amino)-7-methyloct-3-enoic acid (70, Structure A1 (X₃=benzyl), synthesized from Journal of the American Chemical Society, 2005, 127, 12460-12461; 2, and Organic Letter, 2011, 13, 2318-2321) (177 mg, 0.49 mmol) in CH₂Cl₂ (5 mL) at 0° C. was added T₃P (340 mg, 0.53 mmol) and DIEA (106 mg, 0.82 mmol). The reaction mixture was allowed to warm to 25° C. and stirred for 16 h. The reaction mixture was quenched with saturated aqueous NH₄Cl solution (10 mL), and extracted by EtOAc (3×10 mL). The combined organic layer was washed with aqueous HCl (2×10 mL, 1 M), saturated aqueous brine solution (3×10 mL), and dried over Na₂SO₄. The organic layer was concentrated in vacuum and the residue was purified by silica gel column chromatography (petroleum ether/EtOAc=3:1 to 1:1) to give product 56 (308 mg, 89%) as a white foam. MS (ESI): [M+H⁺]=836.5.

Step 6: Synthesis of tert-butyl ((4S,7S,E)-8((S)-2-(((S)-6-amino-1-(isopropylamino)-1-oxohexan-2-yl)carbamoyl)pyrrolidin-1-yl)-7-benzyl-2-methyl-8-oxooct-5-en-4-yl)carbamate (57)

To a solution of compound 56 (308 mg, 0.36 mmol) in CH₂C12 (3 mL) was added DBU (164 mg, 1.08 mmol) at 25° C. and the mixture was stirred for 30 min. Then the solvent was concentrated in vacuum to give product 57 (225 mg, 100%) as a yellow oil. MS (ESI): [M+H⁺]=493.3.

Step 7: Synthesis of 1,4-dimethoxy-2,3,5-trimethylbenzene (59)

To a solution of compound 58 (10 g, 66 mmol) in acetone (100 mL) was added Me₂SO₄ (21.0 g, 165 mmol) and K₂CO₃ (36.0 g, 264 mmol). After the reaction mixture was stirred at 60° C. for 16 hours and then concentrated in vacuum. Saturated aqueous brine solution (200 mL) was added to the flask and the resulting mixture was extracted with EtOAc (3×50 mL). The combined organic layers were washed with water (2×50 mL) and dried over Na₂SO₄. The solvent was concentrated in vacuum and the residue was subjected to silica gel column chromatography (petroleum ether/EtOAc=20:1 to 10:1) to give product 59 (7.2 g, 61%) as a white solid.

Step 8: Synthesis of 2,5-dimethoxy-3,4,6-trimethylbenzaldehyde (60)

The compound 59 (7.2 g, 40 mmol) was dissolved in TFA (50 mL) and then hexamine (6.2 g, 44 mmol) was added to the above solution. The reaction mixture was refluxed under dry conditions for 2 hours. The solvent of TFA was evaporated under reduced pressure, and the residue was dissolved in EtOAc (50 mL) and the organic solution was washed with water (3×50 mL) and then dried over Na₂SO₄. The organic layer was concentrated in vacuum and the residue was subjected to silica gel column chromatography (petroleum ether/EtOAc=20:1 to 10:1) to give product 60 (4.5 g, 54%) as a white solid.

Step 9: Synthesis of ethyl 3-(2,5-dimethoxy-3,4,6-trimethylphenyl)acrylate (61)

To a solution of ethyl 2-(diethoxyphosphoryl) acetate (3.4 g, 15 mmol) in THF (20 mL) was added NaH (600 mg, 15 mmol) at 0° C., after the reaction mixture was stirred at 0° C. for 15 min, then a solution of compound 60 (2.1 g, 10 mmol) in THF (10 mL) was added. The reaction mixture was stirred for 1 h, and then quenched with water (40 mL) and extracted with EtOAc (50 mL). The organic layer was washed with water (2×50 mL), and dried over Na₂SO₄. The organic layer was concentrated in vacuum and the residue was subjected to column chromatography on silica gel (petroleum ether/EtOAc=20:1 to 10:1) to give product 61 (2.2 g, 79%) as a white solid. MS (ESI): [M+H⁺]=278.9.

Step 10: Synthesis of ethyl 3-(2,5-dimethoxy-3,4,6-trimethylphenyl)propanoate (62)

The compound 61 (2.2 g, 7.9 mmol) was dissolved in methanol (20 mL), then Pd/C (50 mg, 10% w/w) was added to the above solution. The reaction mixture was stirred at room temperature in a hydrogen atmosphere for 2 hours. The mixture was filtered and the filtrate was concentrated in vacuum to give product 62 (2.2 g, 100%) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 3.17-3.14 (dd, J=4.0 Hz, 2H), 3.68 (s, 3H), 3.65 (s, 3H), 2.93-2.95 (m, 2H), 2.46-2.49 (m, 2H), 2.23 (s, 3H), 2.20 (s, 6H), 2.25-2.28 (t, J=4.0, 8.0 Hz, 3H).

Step 11: Synthesis of 3-(2,5-dimethoxy-3,4,6-trimethylphenyl)propan-1-ol (63)

The compound 62 (500 mg, 1.8 mmol) was dissolved in THF (10 mL) following by adding LiAlH₄ (80 mg, 2.1 mmol) into the resulting solution at 0° C. The reaction mixture was stirred for 1 h at 0° C. and then sequentially quenched with water (0.81 mL), aqueous NaOH (0.81 mL, 10%) and water (0.81 mL). The mixture was diluted with EtOAc (20 mL) and dried over MgSO₄. The mixture was stirred for 10 min, filtered and the filtrate was concentrated in vacuum to give product 63 (320 mg, 75%) as a yellow oil. MS (ESI): [M+H⁺]=238.9. ¹H NMR (400 MHz, CDCl₃) δ 3.68 (s, 3H), 3.65 (s, 3H), 3.49 (s, 2H), 2.76-2.71 (m, 3H), 2.22 (s, 3H), 2.18 (s, 6H), 1.76-1.74 (m, 2H).

Step 12: Synthesis of 2-(3-hydroxypropyl)-3,5,6-trimethylcyclohexa-2,5-diene-1,4-dione (64)

A solution of compounds 63 (320 mg, 1.3 mmol) in THF (10 mL) was diluted with water (5 mL), and an excess solution of ceric ammonium nitrate (CAN) (1.4 g, 2.6 mmol) in 5 mL water was added at 0° C. The mixture was stirred at room temperature for 2 h. THF was removed under a vacuum and the crude mixture was extracted with three portions of EtOAc (20 mL). The organic layer was washed with saturated aqueous brine solution (2×20 mL) until a water washing reached pH 7.0-7.4, then dried over anhydrous Na₂SO₄ and concentrated in vacuum. The crude products were purified by silica gel column chromatography (petroleum ether/EtOAc=5:1 to 2:1) to give product 64 (210 mg, 78%) as a yellow solid. MS (ESI): [M+H⁺]=208.9. ¹H NMR (400 MHz, CDCl₃) δ 3.58-3.56 (m, 2H), 2.60-2.57 (m, 2H), 2.12 (s, 1H), 2.04-2.01 (m, 9H), 1.70-1.67 (m, 2H).

Step 13: Synthesis of 3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propyl carbonochloridate (65)

To a solution of bis(trichloromethyl) carbonate (324 mg, 1.1 mmol) in toluene (10 mL) was added pyridine (100 mg, 15 mmol) at 0° C. and stirred for 30 min. Then a solution of compound 64 (200 mg, 0.85 mmol) in toluene (2 mL) was added, then the reaction was stirred at 0° C. for 30 min, diluted with water (10 mL) and extracted with EtOAc (10 mL). The organic layer was washed with water (2×20 mL) and dried over Na₂SO₄. The organic layer was concentrated under reduced pressure and the residue was subjected to silica gel column chromatography (petroleum ether/EtOAc=20:1 to 10:1) to give product 65 (140 mg, 61%) as a red oil. MS (ESI): [M+H⁺]=271.2.

Step 14: Synthesis of 3-(2,5-dimethoxy-3,4,6-trimethylphenyl)propanoic acid (66)

A solution of the compound 62 (500 mg, 1.8 mmol) in THF/MeOH (4.0/1.0 mL) was treated with LiOH.H₂O (152 mg, 3.6 mmol, in 0.5 mL water). The reaction mixture was stirred at 25° C. for 2 h, then the solvents were removed in vacuum and the resulting mixture triturated with EtOAc (20 mL) and aqueous HCl (1 M) until pH 3. The organic phase was separated from water phase and the water phase was extracted with EtOAc (2×10 mL). The combined organic layers were washed with saturated aqueous brine solution (2×10 mL), dried over Na₂SO₄, and concentrated in vacuum to afford product 66 (430 mg, 95%), which was used in the next step without purification. MS (ESI): [M+H⁺]=252.9.

Step 15: Synthesis of 3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl) propanoic acid (67)

A solution of compounds 66 (430 mg, 1.7 mmol) in THF (8 mL) was diluted with water (2 mL), and an excess solution of ceric ammonium nitrate (CAN, 1.9 g, 3.4 mmol) in 5 mL water was added at 0° C. The mixture was stirred at room temperature for 2 h. THF was removed under a vacuum and the crude mixture was extracted with three portions of EtOAc (20 mL). The combined organic layers were washed with saturated aqueous brine solution (20 mL) until a water washing reached pH ˜7.0, then dried over anhydrous Na₂SO₄ and concentrated in vacuum. The crude products were purified by a silica-gel column chromatography (petroleum ether/EtOAc=5:1 to 2:1) to give product 67 (300 mg, 80%) as a red oil. MS (ESI): [M+H⁺]=223.3.

Step 16: Synthesis of tert-butyl ((4S,7S,E)-7-benzyl-8-((S)-2-(((S)-6-(3-(4,5-dimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propanamido)-1-(isopropylamino)-1-oxohexan-2-yl)carbamoyl)pyrrolidin-1-yl)-2-methyl-8-oxooct-5-en-4-yl)carbamate (I)

To a solution of compound 57 (150 mg, 0.24 mmol) and 67 (59 mg, 0.26 mmol) in CH₂C12 (5 mL) was added T₃P (198 mg, 0.31 mmol) and DIEA (40 mg, 0.31 mmol) at 0° C. The reaction mixture was allowed to warm to 25° C. and stirred for 2 h. The reaction mixture was quenched with saturated aqueous NH₄Cl solution, and extracted by EtOAc (3×10 mL). The combined organic layer was washed with aqueous (5%) NaHCO₃, saturated aqueous brine solution (3×10 mL) and dried over Na₂SO₄. After concentration under reduced pressure, the residue was purified by prep-HPLC (Venusil XBP C18(5 μm, 30×250 mm), 0.1% HCl in eluant) to give product I (40 mg, 20%) as a yellow solid. MS (ESI): [M+H⁺]=832.5. ¹H NMR (400 MHz, CDCl₃) δ 7.24-7.12 (m, 5H), 6.74-6.73 (m, 1H), 6.32-6.39 (m, 2H), 5.41-5.55 (m, 2H), 5.06 (s, 1H), 4.39-4.41 (m, 2H), 4.06-4.08 (m, 2H), 3.19-3.39 (m, 6H), 2.76-2.79 (m, 3H), 2.28-2.29 (m, 2H), 1.18-2.05 (m, 37H), 0.85-0.87 (m, 6H).

Step 17: Synthesis of tert-butyl N-[(4S,5E,7S)-7-benzyl-8-[(2S)-2-{[(1S)-5-({[3-(4,5-dimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propoxy]carbonyl}amino)-1-[(propan-2-yl) carbamoyl]pentyl]carbamoyl}pyrrolidin-1-yl]-2-methyl-8-oxooct-5-en-4-yl]carbamate (II)

To a solution of compound 57 (320 mg, 0.51 mmol) and compound 65 (165 mg, 0.61 mmol) in CH₂C12 (5 mL) was added DIEA (79 mg, 0.61 mmol) at 0° C. The reaction mixture was allowed to warm to 25° C. and stirred for 2 h. The reaction mixture was quenched with saturated aqueous NH₄Cl solution (20 mL), and extracted by EtOAc (3×10 mL). The combined organic layer was washed with saturated aqueous brine solution (3×10 mL) and dried over Na₂SO₄. The residue after concentration under reduced pressure was purified by prep-HPLC (Venusil XBP C18(5 um, 30×250 mm), 0.1% HCl in eluant) to give product II (70 mg, 16%) as a yellow solid. MS (ESI): [M+H⁺]=862.6. ¹H NMR (400 MHz, CDCl₃) δ 7.24-7.12 (m, 5H), 6.8-6.70 (m, 1H), 6.25-6.26 (m, 1H), 5.39-5.57 (m, 2H), 5.03 (s, 1H), 4.40-4.41 (m, 2H), 4.01-4.07 (m, 3H), 3.11-3.38 (m, 5H), 2.50-2.54 (m, 2H), 1.17-2.00 (m, 43H), 0.84-0.87 (m, 6H).

Step 18: Synthesis of tert-butyl N-[(4S,5E,7S)-7-benzyl-8-[(2S)-2-{[(1S)-5-[({[10-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)decyl]oxy}carbonyl)amino]-1-[(propan-2-yl)carbamoyl]pentyl]carbamoyl}pyrrolidin-1-yl]-2-methyl-8-oxooct-5-en-4-yl]carbamate (III)

To a solution of compound 57 (110 mg, 0.18 mmol) and compound 69 (77 mg, 0.2 mmol, synthesized by methods analog for compound 65) in CH₂Cl₂ (5 mL) was added DIEA (47 mg, 0.36 mmol) at 0° C. The reaction mixture was allowed to warm to 25° C. and stirred for 2 h. The reaction mixture was quenched with saturated aqueous NH₄Cl solution (20 mL), and extracted by EtOAc (3×10 mL). The combined organic layer was washed with saturated aqueous brine (3×10 mL) solution and dried over Na₂SO₄. The residue after concentration under reduced pressure and was purified by prep-HPLC (Venusil XBP C18(5 μm, 30×250 mm), 0.1% HCl in eluant) to give product III (40 mg, 22%) as a yellow solid. MS (ESI): [M+H]=992.7. ¹H NMR (400 MHz, CDCl₃) δ 7.24-7.12 (m, 5H), 6.71-6.69 (m, 1H), 6.30-6.28 (m, 1H), 5.58-5.57 (m, 1H), 5.44-5.39 (m, 1H), 5.07 (s, 1H), 4.41-4.40 (m, 2H), 4.07-3.97 (m, 9H), 3.38-3.11 (m, 6H), 2.72-2.68 (m, 2H), 2.44-2.43 (m, 2H), 2.02-1.17 (m, 50H), 0.86-0.84 (m, 6H).

Example 2: tert-Butyl ((4S,7S,E)-7-benzyl-8-((S)-2-(((S)-6-(3-(4,5-dimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propanamido)-1-(isopropylamino)-1-oxohexan-2-yl)carbamoyl)pyrrolidin-1-yl)-2-methyl-8-oxooct-5-en-4-yl) carbamate (IV) and Synthesis of tert-butyl N-[(4S,5E,7S)-7-benzyl-8-[(2S)-2-{[(1S)-5-({[3-(4,5-dimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propoxy]carbonyl}amino)-1-[(propan-2-yl) carbamoyl]pentyl]carbamoyl}pyrrolidin-1-yl]-2-methyl-8-oxooct-5-en-4-yl]carbamate (V)

Step 1: Synthesis of 1,4-dimethoxy-2,3-dimethylbenzene (72)

To a solution of compound 71 (5 g, 36.2 mmol) in acetone (100 mL) was added Me₂SO₄ (18.2 g, 144.8 mmol) and K₂CO₃ (25 g, 181 mmol). After the reaction mixture was stirred at 60° C. for 16 hours and then the solvent was concentrated in vacuum. Saturated aqueous brine solution (200 mL) was added to the flask and the resulting mixture was extracted with EtOAc (3×50 mL). The combined organic layers were washed with water (2×50 mL) and dried over Na₂SO₄. The solvent was evaporated under reduced pressure and the residue was subjected to silica gel column chromatography (petroleum ether/EtOAc=20:1 to 10:1) to give product 72 (5.5 g, 92%) as a white solid.

Step 2: Synthesis of 2,5-dimethoxy-3,4-dimethylbenzaldehyde (73)

The compound 72 (5 g, 30.1 mmol) was dissolved in TFA (50 mL) and hexamine (4.7 g, 33.1 mmol) was added to the resulting solution. The reaction mixture was refluxed under dry conditions for 2 hours. The TFA was evaporated under reduced pressure and the residue was dissolved in EtOAc (100 mL). and the resulting organic solution was washed with water (3×50 mL) and then dried over Na₂SO₄. The solvent organic phase was evaporated concentrated in vacuum and the residue was subjected to silica gel column chromatography (petroleum ether/EtOAc=20:1 to 10:1) to give product 73 (3.1 g, 53%) as a white solid.

Step 3: Synthesis of ethyl 3-(2,5-dimethoxy-3,4-dimethylphenyl)acrylate (74)

To a solution of ethyl 2-(diethoxyphosphoryl)acetate (3.1 g, 16 mmol) in THF (20 mL) was added NaH (660 mg, 16 mmol) at 0° C., and the reaction mixture was stirred at 0° C. for 15 min, and then a solution of 73 (2.2 g, 10.2 mmol) in THF (10 mL) was added. The reaction mixture was stirred for 10 min, water (40 mL) was added and the result was extracted with EtOAc (50 mL). The organic layer was washed with water (2×50 ml) and dried over Na₂SO₄. The solvent in the organic layer was evaporated under reduced pressure and the residue was subjected to silica gel column chromatography (petroleum ether/EtOAc=20:1 to 10:1) to give product 74 (3.4 g, 81%) as a white solid.

Step 4: Synthesis of ethyl 3-(2,5-dimethoxy-3,4-dimethylphenyl)propanoate (75)

The compound 74 (3.4 g, 7.9 mmol) was dissolved in methanol (20 mL) and then Pd/C (50 mg, 10% w/w) was added to the above solution. The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 2 hours. The mixture was filtered and the filtrate was concentrated in vacuum to give product 75 (3.4 g, 100%) as a yellow oil. MS (ESI): [M+H]=266.9.

Step 5: Synthesis of 3-(2,5-dimethoxy-3,4-dimethylphenyl)propan-1-ol (76)

The compound 75 (500 mg, 1.9 mmol) was dissolved in THF (10 mL) and then LiAlH₄ (80 mg, 2.1 mmol) was added to the resulting solution at 0° C. The reaction mixture was stirred for 1 h at 0° C. and then sequentially quenched with water (0.81 mL), aqueous NaOH (0.81 mL, 10%) and water (0.81 mL). The mixture was added diluted with EtOAc (10 mL) and dried over MgSO₄. The mixture was stirred for 10 min and filtered, and the filtrate was concentrated in vacuum to give product 76 (350 mg, 82%) as a yellow oil.

Step 6: Synthesis of 5-(3-hydroxypropyl)-2,3-dimethylcyclohexa-2,5-diene-1,4-dione (77)

A solution of compounds 76 (350 mg, 1.6 mmol) in THF (10 mL) was diluted with water (5 mL), and an excess solution of ceric ammonium nitrate (CAN, 1.8 g, 3.2 mmol) in 5 mL water was added at 0° C. The mixture was stirred at room temperature for 2 h. The progress of the reaction was monitored by TLC. After completion, THF was removed under a vacuum and the crude mixture was extracted with three portions of EtOAc (3×20 mL). The combined organic layers were washed with saturated aqueous brine solution, then dried over anhydrous Na₂SO₄ and concentrated in vacuum. The crude products were purified by silica-gel column chromatography (petroleum ether/EtOAc=5:1 to 2:1) to give product 77 (210 mg, 68%) as a yellow solid. MS (ESI): [M+H⁺]=194.8.

Step 7: Synthesis of 3-(4,5-dimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propyl carbonochloridate (78)

To a solution of bis(trichloromethyl) carbonate (210 mg, 1.1 mmol) in toluene (10 mL) was added pyridine (100 mg, 15 mmol) at 0° C. and stirred for 30 min. Then a solution of compound 7 (200 mg, 0.85 mmol) in toluene (2 mL) was added to the reaction at 0° C. The reaction mixture was stirred at 0° C. for 30 min, diluted with water (40 mL) and extracted with EtOAc (50 mL). The organic layer was washed with water (2×50 mL) and dried over Na₂SO₄. The organic layer was concentrated in vacuum and the residue was subjected to silica gel column chromatography (petroleum ether/EtOAc=20:1 to 10:1) to give product 78 (140 mg, 50%) as a red oil. MS (ESI): [M+H⁺]=256.8.

Step 8: Synthesis of 3-(2,5-dimethoxy-3,4-dimethylphenyl)propanoic acid (79)

A solution of the compound 75 (500 mg, 1.9 mmol) in THF/MeOH (4.0/1.0 mL) was treated with LiOH.H₂O (152 mg, 3.8 mmol, in 0.5 mL water) at 0° C. Then the reaction mixture was stirred at 25° C. for 2 h, and the solvents were concentrated in vacuum and the resulting mixture was triturated with EtOAc (20 mL) and aqueous HCl (1 M) until pH 3. The organic phase was separated from water phase. The water phase was extracted with EtOAc (2×10 mL), and the combined organic layers were washed with saturated aqueous brine solution (2×10 mL), dried over Na₂SO₄, and concentrated in vacuum to afford product 79 (380 mg, 84%), which was carried on crude to use in the next step without purification. MS (ESI): [M+H⁺]=238.9.

Step 9: Synthesis of 3-(4,5-dimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propanoic acid (80)

A solution of compounds 79 (380 mg, 1.6 mmol) in THF (8 mL) was diluted with water (2 mL), and an excess solution of ceric ammonium nitrate (CAN) (1.9 g, 3.4 mmol) in 5 mL water was added at 0° C. The mixture was stirred at room temperature for 2 h. The progress of the reaction was monitored by TLC. After completion, THF was removed under vacuum and the crude residue was extracted with three portions of EtOAc (3×20 mL). The combined organic layers were washed with saturated aqueous brine solution (20 mL), then dried over anhydrous Na₂SO₄ and concentrated in vacuum. The crude products were purified by a silica gel column chromatography (petroleum ether/EtOAc=5:1 to 2:1) to give product 80 (110 mg, 33%) as a red oil. MS (ESI): [M+H⁺]=208.8.

Step 10: Synthesis of tert-butyl ((4S,7S,E)-7-benzyl-8-((S)-2-(((S)-6-(3-(4,5-dimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propanamido)-1-(isopropylamino)-1-oxohexan-2-yl)carbamoyl)pyrrolidin-1-yl)-2-methyl-8-oxooct-5-en-4-yl) carbamate (IV)

To a solution of compound 57 (150 mg, 0.24 mmol) and compound 80 (54 mg, 0.26 mmol) in CH₂Cl₂ (5 mL) was added T₃P (198 mg, 0.31 mmol) and DIEA (40 mg, 0.31 mmol) at 0° C. The reaction mixture was allowed to warm to 25° C. and stirred for 2 h. The reaction mixture was quenched with saturated aqueous NH₄Cl solution (20 mL), and extracted with EtOAc (3×10 mL). The organic layer was washed with aqueous (5%) NaHCO₃(20 mL), brine (3×10 mL) and dried over Na₂SO₄. The residue was evaporated under reduced pressure and purified by prep-HPLC (Venusil XBP C18(5 μm, 30×250 mm), 0.1% HCl in eluant) to give product IV (42 mg, 21%) as a yellow solid. MS (ESI): [M+H⁺]=818.6. ¹H NMR (400 MHz, CDCl₃) δ 7.24-7.00 (m, 6H), 6.64 (s, 1H), 6.33-6.31 (m, 1H), 5.62-5.56 (m, 1H), 5.44-5.41 (m, 1H), 4.80 (s, 1H), 4.42-4.40 (m, 2H), 4.08-4.05 (m, 2H), 3.41-3.07 (m, 5H), 2.77-2.41 (m, 3H), 2.07-1.17 (m, 37H), 0.88-0.86 (m, 6H).

Step 11: Synthesis of tert-butyl N-[(4S,5E,7S)-7-benzyl-8-[(2S)-2-{[(1S)-5-({[3-(4,5-dimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propoxy]carbonyl}amino)-1-[(propan-2-yl) carbamoyl]pentyl]carbamoyl}pyrrolidin-1-yl]-2-methyl-8-oxooct-5-en-4-yl]carbamate (V)

To a solution of compound 67 (320 mg, 0.51 mmol) and compound 88 (156 mg, 0.61 mmol) in CH₂C12 (5 mL) was added DIEA (79 mg, 0.61 mmol) at 0° C. The reaction mixture was allowed to warm to 25° C. and stirred for 2 h. The reaction mixture was quenched with saturated NH₄Cl solution, and extracted by EtOAc (3×10 mL). The organic layer was washed with brine (3×10 mL) and dried over Na₂SO₄. The residue was concentrated in vacuum and purified by pre-HPLC (Venusil XBP C18(5 μm, 30×250 mm), 0.1% HCl in eluant) to give product V (45 mg, 10%) as a yellow solid. MS (ESI): [M+H+]=848.5. ¹H NMR (400 MHz, CDCl₃) δ 7.24-7.13 (m, 5H), 6.77-6.48 (m, 2H), 5.62-5.56 (m, 1H), 5.44-5.41 (m, 1H), 5.39-5.20 (m, 1H), 4.41-4.33 (m, 2H), 4.08-4.05 (m, 3H), 3.73-3.11 (m, 7H), 2.79-2.47 (m, 2H), 2.01-1.17 (m, 39H), 0.87-0.85 (m, 6H).

Example 3: tert-Butyl ((4S,7S,E)-7-benzyl-8-(S)-2-(((S)-1-(isopropylamino)-1-oxo-6-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)hexan-2-yl)car-bamoyl)pyrrolidin-1-yl)-2-methyl-8-oxooct-5-en-4-yl)carbamate (VI)

Step 1: Synthesis of (S)-1-benzyl 2-tert-butyl 5-oxopyrrolidine-1,2-dicarboxylate (92)

To a solution of compound 91 (1 g, 5.4 mmol) in THF (20 mL) was added NaH (238 mg, 5.95 mmol, 60% in oil), then the mixture was stirred at 25° for 30 min, subsequently CbzCl (1.02 g, 5.95 mmol) was added to the above mixture and the stirring was continued for further 20 h. The solution was concentrated in vacuum, saturated aqueous NH₄Cl solution (100 mL) was added to the residue and extracted with EtOAc (2×100 mL). The combined organic phase was dried over MgSO₄, filtered and concentrated in vacuum. The crude product was purified on silica gel column (petroleum ether/EtOAc=3:1) to obtain the titled compound 92 (1 g, 58%) as a colorless oil. MS (ESI): [M+H⁺]=319.9.

Step 2: Synthesis of (S)-tert-butyl 2-(((benzyloxy)carbonyl)amino)-5-hydroxypentanoate (93)

To a solution of compound 92 (1 g, 3.13 mmol), KH₂PO₄ (3.2 g, 23.5 mmol) in MeOH (25 mL) and H₂O (5 mL) was added NaBH₄ (893 mg, 23.5 mmol). The mixture was stirred at 25° C. for 1 h, then organic solution was concentrated in vacuum. To the residue was added EtOAc (100 mL) and washed with H₂O (3×50 mL). The organic phase was dried over MgSO₄, filtered and concentrated in vacuum to obtain the titled compound 93 (1 g, 100%) as a colorless oil. MS (ESI): [M+H⁺]=324.0.

Step 3: Synthesis of (S)-tert-butyl 5-(benzo[d]thiazol-2-ylthio)-2-(((benzyloxy)carbonyl)amino)pentanoate (94)

To a solution of compound 93 (1 g, 3.13 mmol), benzo[d]thiazole-2-thiol (620 mg, 3.7 mmol), Ph₃P (969 mg, 3.7 mmol) in THF (30 mL) at 0° C. was added DIAD (747 mg, 3.7 mmol). The mixture was stirred at 0° C. for 3 h, then organic solution was concentrated in vacuum. To the residue was added EtOAc (100 mL) and washed with H₂O (3×50 mL). The organic phase was concentrated and purified on silica gel column (petroleum ether/EtOAc=5:1) to obtain the titled compound 94 (1.33 g, 90%) as a colorless oil. MS (ESI): [M+H⁺]=473.1.

Step 4: Synthesis of (S)-tert-butyl 5-(benzo[d]thiazol-2-ylsulfonyl)-2-(((benzyloxy)carbonyl)amino)pentanoate (95)

To a solution of compound 94 (1.33 g, 2.8 mmol) in CH₂Cl₂ (30 mL) was added m-CPBA (1.07 g, 6.2 mmol). The mixture was stirred at 25° C. for 1 h, then organic solution was concentrated in vacuum. To the residue was added EtOAc (100 mL) and washed with 2N Na₂CO₃ in water (3×50 mL). The organic phase was concentrated and purified by silica gel column chromatography (petroleum ether/EtOAc=5:1) to obtain the titled compound 95 (930 mg, 66%) as a colorless oil. MS (ESI): [M+H⁺]=505.1.

Step 5: synthesis of (S,Z)-tert-butyl 2-(((benzyloxy)carbonyl)amino)-6-(2,5-dimethoxy-3,4,6-trimethylphenyl)hex-5-enoate (96)

To a solution of compound 95 (930 mg, 1.85 mmol) in THF (25 mL) at −78° C. was added KHMDS (6.1 mL, 6.1 mmol, 1M in THF) under N₂ atmosphere. The mixture was stirred at −78° C. for 30 min, then 2,5-dimethoxy-3,4,6-trimethylbenzaldehyde (384 mg, 1.85 mmol) in THF (5 mL) was added and stirred at −78° C. for 3 h, then warmed to 0° C. and stirred for another 0.5 h. The mixture was quenched with H₂O (50 mL) and extracted with EtOAc (50 mL). The organic phase was washed with brine (3×50 mL), concentrated and purified by silica gel column chromatography (petroleum ether/EtOAc=5:1) to obtain the titled compound 96 (443 mg, 48%) as an orange oil. MS (ESI): [M+H⁺]=498.2.

Step 6: Synthesis of (S,Z)-2-(((benzyloxy)carbonyl)amino)-6-(2,5-dimethoxy-3,4,6-trimethylphenyl)hex-5-enoic acid (97)

To a solution of compound 96 (443 mg, 0.89 mmol) in CH₂C12 (25 mL) was added TFA (1 mL). The mixture was stirred at 25° C. for 18 h, then concentrated in vacuum to obtain the titled compound 97 (393 mg, 100%) as a yellow solid. MS (ESI): [M+H⁺]=442.0.

Step 7: Synthesis of (S,Z)-benzyl (6-(2,5-dimethoxy-3,4,6-trimethylphenyl)-1-(isopropylamino)-1-oxohex-5-en-2-yl)carbamate (98)

To a solution of compound 97 (393 mg, 0.89 mmol), propan-2-amine (158 mg, 2.7 mmol), DIEA (344 mg, 2.7 mmol) in CH₂Cl₂ (20 mL) was added T₃P (736 mg, 1.2 mmol). The mixture was stirred at 25° C. for 12 h, then the organic solution was concentrated in vacuum. To the residue was added EtOAc (100 mL) and washed with H₂O (3×50 mL). The organic phase was collected and concentrated in vacuum to obtain the titled compound 98 (430 mg, 100%) as a yellow solid. MS (ESI): [M+H⁺]=483.1.

Step 8: Synthesis of (S)-2-amino-6-(2,5-dimethoxy-3,4,6-trimethylphenyl)-N-isopropylhexanamide (99)

To a solution of compound 98 (430 mg, 0.89 mmol) in MeOH (10 mL) was added Pd/C (50 mg, 10%). The mixture was stirred at 25° C. under H₂ atmosphere for 12 h, filtered, and the solids were washed with MeOH (5 mL). The organic filtrates were combined and concentrated in vacuum to obtain the titled compound 99 (311 mg, 100%) as a yellow solid. MS (ESI): [M+H⁺]=351.0.

Step 9: synthesis of (S)-tert-butyl (6-(2,5-dimethoxy-3,4,6-trimethylphenyl)-1-(isopropylamino)-1-oxohexan-2-yl)carbamate (100)

To a solution of compound 99 (311 mg, 0.89 mmol), Et₃N (180 mg, 1.78 mmol) in CH₂C12 (15 mL) was added Boc₂O (215 mg, 0.98 mmol). The mixture was stirred at 25° C. for 3 h, then the organic solution was concentrated. To the residue was added EtOAc (100 mL) and washed with H₂O (3×50 mL). The organic phase was collected and concentrated to obtain the titled compound 100 (401 mg, 100%) as a yellow solid. MS (ESI): [M+H⁺]=451.2.

Step 10: Synthesis of (S)-tert-butyl (1-(isopropylamino)-1-oxo-6-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)hexan-2-yl)carbamate (101)

To a solution of compound 100 (401 mg, 0.89 mmol) in CH₃CN (10 mL) and H₂O (10 mL) was added CAN (975 mg, 1.78 mmol). The mixture was stirred at 25° C. for 3 h, then organic solution was concentrated in vacuum. To the residue was added EtOAc (100 mL) and washed with H₂O (3×50 mL). The organic phase was concentrated and purified by silica gel column chromatography (petroleum ether/EtOAc=3:1) to obtain the titled compound 101 (104 mg, 28%) as a yellow solid. MS (ESI): [M+H⁺]=421.1.

Step 11: synthesis of (S)-2-amino-N-isopropyl-6-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)hexanamide (102)

To the compound 101 (104 mg, 0.25 mmol) EtOAc (5 mL) was added HCl in MeOH (5 mL, 3 M) and then the mixture was stirred at 25° C. for 1 h. The organic solution was concentrated in vacuum to obtain the titled compound 102 (79 mg, 100%) as a yellow solid. MS (ESI): [M+H⁺]=321.0.

Step 12: Synthesis of (S)-tert-butyl 2-(((S)-1-(isopropylamino)-1-oxo-6-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)hexan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (103)

To a solution of compound 102 (79 mg, 0.25 mmol), (S)-1-(tert-butoxycarbonyl) pyrrolidine-2-carboxylic acid (81 mg, 0.38 mmol), DIEA (97 mg, 0.75 mmol) in CH₂C12 (10 mL) was added T3P (206 mg, 0.33 mmol). The mixture was stirred at 25° C. for 12 h and then the mixture was concentrated in vacuum. To the residue was added EtOAc (100 mL) and washed with H₂O (3×50 mL). The organic phase was collected and concentrated in vacuum to obtain the titled compound 103 (129 mg, 100%) as a yellow solid. MS (ESI): [M+H⁺]=510.2.

Step 13: Synthesis of (S)—N-((S)-1-(isopropylamino)-1-oxo-6-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)hexan-2-yl)pyrrolidine-2-carboxamide (104)

To the compound 103 (129 mg, 0.25 mmol) was added HCl in MeOH (3 mL, 3 M) and EtOAc (3 mL) and then the mixture was stirred at 25° C. for 1 h. The organic solution was concentrated in vacuum to obtain the titled compound 104 (104 mg, 100%) as a yellow solid. MS (ESI): [M+H⁺]=418.1.

Step 14: Synthesis of tert-butyl ((4S,7S,E)-7-benzyl-8-((S)-2-(((S)-1-(isopropylamino)-1-oxo-6-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)hexan-2-yl)carbamoyl)pyrrolidin-1-yl)-2-methyl-8-oxooct-5-en-4-yl)carbamate (VI)

To a solution of compound 104 (104 mg, 0.25 mmol), (2S,5S,E)-2-benzyl-5-((tert-butoxycarbonyl)amino)-7-methyloct-3-enoic acid (70, 90 mg, 0.25 mmol), DIEA (97 mg, 0.75 mmol) in CH₂Cl₂ (10 mL) was added T₃P (206 mg, 0.33 mmol). The mixture was stirred at 25° C. for 2 h and concentrated in vacuum. To the residue was added EtOAc (50 mL) and washed with H₂O (3×50 mL). The organic phase was concentrated and purified by prep-HPLC (Durashell C18 (10 um, 30×250 mm), 0.5% NH₃H₂O in eluant) to obtain the titled compound VI (52 mg, 27%) as a yellow solid. MS (ESI): [M+H⁺]=761.5. NMR (400 MHz, CDCl₃) δ 7.26-7.24 (m, 2H), 7.14-7.13 (m, 2H), 6.64 (d, 1H), 6.37 (d, 1H), 5.61-5.57 (m, 1H), 5.46-5.43 (m, 1H), 5.24 (d, 1H), 4.42 (s, 1H), 4.32 (s, 1H), 4.08-4.06 (m, 2H), 3.56 (s, 1H), 3.38-3.37 (m, 1H), 3.23-3.22 (m, 1H), 3.16-3.12 (m, 1H), 2.78-2.75 (m, 1H), 2.44-2.39 (m, 2H), 1.98-1.85 (m, 16H), 1.50 (s, 1H), 1.41-1.24 (m, 12H), 1.21-1.18 (m, 8H), 0.86-0.83 (m, 6H).

Example 4: ethyl ((4S,7S,E)-7-benzyl-8-((S)-2-(((S)-1-(isopropylamino)-1-oxo-6-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)hexan-2-yl)carbamoyl)pyrrolidin1-yl)-2-methyl-8-oxooct-5-en-4-yl)carbamate (VII)

Step 1: Synthesis of (S)-1-((2S,5S,E)-5-amino-2-benzyl-7-methyloct-3-enoyl)-N-((S)-1-(isopropylamino)-1-oxo-6-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)hexan-2-yl)pyrrolidine-2-carboxamide (108)

To a 50-mL single-neck flask was added the compound VI (15 mg, 0.02 mmol) and HCl in MeOH (5 mL, 3N), then the mixture was stirred at 25° C. for 1 h. The organic solution was concentrated in vacuum to obtain the titled compound 108 (13 mg, 100%). MS (ESI): [M+H⁺]=661.3.

Step 2: Synthesis of ethyl ((4S,7S,E)-7-benzyl-8-((S)-2-(((S)-1-(isopropylamino)-1-oxo-6-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)hexan-2-yl)carbamoyl)pyrroli-dinl-yl)-2-methyl-8-oxooct-5-en-4-yl)carbamate (VII)

To a solution of 108 (13 mg, 0.02 mmol) and DIEA (5 mg, 0.04 mmol) in DCM (5 mL) was added ethyl chloroformate (2 mg, 0.02 mmol), then the reaction was stirred at 25° C. for 0.5 h. To the mixture was added DCM (50 mL) and washed with H₂O (3×50 mL). The organic phase was concentrated in vacuum and purified by prep-HPLC (Durashell C18(10 um, 30×250 mm), 0.5% NH₃H₂O in eluant) to obtain the titled compound VII (9 mg, 60%) as a yellow solid. MS (ESI): [M+H⁺]=733.4. ¹H NMR (400 MHz, CDCl₃) δ 7.23-7.13 (m, 5H), 5.63-5.57 (m, 1H), 5.43-5.33 (m, 2H), 4.44-4.33 (m, 1H), 4.14-4.04 (m, 3H), 3.56-3.12 (m, 4H), 2.79-2.74 (m, 1H), 2.45-2.38 (m, 1H), 2.08-2.01 (m, 1H), 1.99 (s, 9H), 1.94-1.84 (m, 2H), 1.52-1.15 (m, 24H), 0.87-0.84 (m, 6H).

Example 5: tert-butyl N-[(4S,5E,7S)-7-benzyl-8-[(2S)-2-{[(1S)-1-{[(1S)-4-({[3-(2,5-dimethoxy-3,4,6-trimethylphenyl)propoxy]carbonyl}amino)-1-[(propan-2-yl)carbamoyl]butyl]carbamoyl}-2-methylpropyl]carbamoyl}pyrrolidin-1-yl]-2-methyl-8-oxooct-5-en-4-yl]carbamate (VIII)

Step 1: Synthesis of tert-butyl N-[(4S,5E,7S)-7-benzyl-8-[(2S)-2-{[(1S)-1-{[(1S)-4-({[3-(2,5-dimethoxy-3,4,6-trimethylphenyl)propoxy]carbonyl}amino)-1-[(propan-2-yl)carbamoyl]-butyl]carbamoyl}-2-methylpropyl]carbamoyl}pyrrolidin-1-yl]-2-methyl-8-oxooct-5-en-4-yl]carbamate (VIII)

To a solution of 56 (160 mg, 0.17 mmol) in DCM (5 mL) was added DBU (130 mg, 0.86 mmol). The reaction was stirred at 25° C. for 15 min, then 3-(2,5-dimethoxy-3,4,6-trimethylphenyl)propyl carbonochloridate (110, 56 mg, 0.19 mmol) was added and the mixture was stirred at 25° C. for 30 min. To the mixture was added DCM (50 mL) and washed with H₂O (3×50 mL). The organic phase was concentrated in vacuum and purified by prep-HPLC (Durashell C18(10 μm, 30×250 mm), 0.5% NH₃H₂O in eluant) to obtain the titled compound VIII (20 mg, 12%) as a white solid. MS (ESI): [M+H]=977.7. ¹H NMR (400 MHz, CDCl₃) δ 7.23-7.13 (m, 5H), 6.82 (d, 1H), 6.53 (d, 1H), 5.63-5.34 (m, 2H), 4.90-4.69 (m, 1H), 4.42-4.03 (m, 6H), 3.62 (s, 6H), 3.60-3.14 (m, 6H), 2.76-2.62 (m, 3H), 2.30-2.26 (m, 1H), 2.16 (s, 9H), 2.04-0.84 (m, 43H).

Example 6: Cyclopropylmethyl N-[(4S,5E,7S)-7-benzyl-2-methyl-8-oxo-8-[(2S)-2-{[(1S)-1-[(propan-2-yl)carbamoyl]-5-({[3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl) propoxy]carbonyl}amino)pentyl]carbamoyl}pyrrolidin-1-yl]oct-5-en-4-yl]carbamate (IX)

Step 1: Synthesis of cyclopropylmethyl carbonochloridate (112)

To a solution of bis(trichloromethyl) carbonate (10.6 g, 36.1 mmol) in toluene (50 mL) was added pyridine (2.6 g, 33.4 mmol) at 0° C., and the mixture was stirred for 30 min. Then a solution of 111 (2.0 g, 27.8 mmol) in toluene (5 mL) was added. The reaction was stirred for 30 min, water (40 mL) was added and extracted with EA (50 mL). The organic layers were washed with water (2×50 mL) and dried over Na₂SO₄. The solvent was concentrated in vacuum and the residue was subjected to column chromatography on silica gel (petroleum ether/EtOAc=20:1 to 10:1) to give the titled compound 112 (2.9 g, 78%) as an oil.

Step 2: Synthesis of 3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propyl ((S)-5-((S)-1-((2S,5S,E)-5-amino-2-benzyl-7-methyloct-3-enoyl)pyrrolidine-2-carboxamido)-6-(isopropylamino)-6-oxohexyl)carbamate (113)

A solution of compound IV (20 mg, 0.02 mmol) in HCl in MeOH (5 mL, 4 N) was stirred at 25° C. for 2 h and then the solvent was concentrated in vacuum to give the titled product 113 (15 mg, 100%) as a white foam. MS (ESI): [M+H⁺]=762.4.

Step 3: Synthesis of cyclopropylmethyl N-[(4S,5E,7S)-7-benzyl-2-methyl-8-oxo-8-[(2S)-2-{[(1S)-1-[(propan-2-yl)carbamoyl]-5-({[3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl) propoxy]carbonyl}amino)pentyl]carbamoyl}pyrrolidin-1-yl]oct-5-en-4-yl]carbamate (IX)

To a solution of compound 113 (15 mg, 0.02 mmol) and compound 112 (4 mg, 0.24 mmol) in DCM (5 mL) was added DIEA (5 mg, 0.04 mmol) at 0° C. The reaction mixture was allowed to warm to 25° C. and stirred for 2 h. The reaction mixture was quenched with saturated NH₄Cl solution (10 mL), and extracted by EA (3×10 mL). The organic layer was washed with aqueous brine (20 mL) and dried over Na₂SO₄. The residues were concentrated under reduced pressure and purified by prep-HPLC (Durashell C18(10 μm, 30×250 mm), 0.1% NH₃.H₂O in eluant) to give the product IX (9 mg, 53%) as a yellow solid. MS (ESI): [M+H⁺]=860.6. ¹H NMR (400 MHz, CDCl₃) δ 7.23-7.13 (m, 5H), 6.78-6.76 (m, 1H), 6.30-6.29 (m, 1H), 5.60-5.33 (m, 2H), 4.42-3.81 (s, 4H), 3.58-3.13 (m, 4H), 2.72-2.68 (m, 1H), 2.55-2.53 (m, 1H), 2.01-1.83 (m, 40H), 0.88-0.86 (m, 6H), 0.542-0.52 (m, 2H), 0.25-0.24 (m, 2H).

Example 7: tert-Butyl ((4S,7S,E)-7-benzyl-8-((S)-2-(((S)-1-(isopropylamino)-1-oxo-3-(4-(3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propoxy)phenyl)propan-2-yl)carbamoyl)pyrrolidin-1-yl)-2-methyl-8-oxooct-5-en-4-yl)carbamate (X)

Step 1: Synthesis of (S)-2-((tert-butoxycarbonyl)amino)-3-(4-hydroxyphenyl) propanoic acid (122)

To a solution of compound 121 (2.0 g, 11.0 mmol) in dioxane/H₂O (20/10 mL) was added Na₂CO₃ (3.5 g, 33.0 mmol) and Boc₂O (3.5 g, 16.5 mmol) at 0° C. The reaction mixture was allowed to warm to 25° C. and stirred for 16 h and then was removed under vacuum. Water (20 mL) was added to the mixture, and extracted by EA (10 mL). The water layer was added HCl (4M in water) until pH=2-3 and extracted by EA (3×10 mL). The combined organic layer was washed with aqueous brine (20 mL), dried over Na₂SO₄ and concentrated in vacuum to give the titled product 122 (2.8 g, 90%) as a yellow solid. MS (ESI): [M+H⁺]=281.9.

Step 2: Synthesis of (S)-tert-butyl (3-(4-hydroxyphenyl)-1-(isopropylamino)-1-oxopropan-2-yl) carbamate (123)

To the solution of compound 122 (2.8 g, 9.9 mmol) and propan-2-amine (0.88 g, 14.9 mmol) in DCM (20 mL) at 0° C. was added DIEA (2.6 g, 19.8 mmol), T₃P (50% in EA, 9.4 g, 14.9 mmol) was added slowly, and the reaction mixture was allowed to warm to 25° C. and stirred for 18 h. The reaction mixture was washed with 5% aqueous Na₂CO₃ (10 mL) and water (20 mL), and the organic layer was dried over MgSO₄ and concentrated in vacuum. The residual semisolids were dried under vacuum to obtain a solid product 123 (2.8 g, 88%). MS (ESI): [M+H⁺]=322.9.

Step 3: Synthesis of 3-(2,5-dimethoxy-3,4,6-trimethylphenyl)propyl 4-methylbenzenesulfonate (129)

To the solution of compound 110 (3.0 g, 12.6 mmol) in 10 mL DMF was added tosyl chloride (2.87 g, 15.1 mmol), and the mixture was stirred at 0° C. for 10 min. Then, DIEA (1.9 g, 15.1 mmol) was added slowly to the mixture, the reaction was stirred at 25° C. for 5 h, TLC was used to monitor the reaction progress. The reaction was quenched by water (10 mL), extracted by EA (3×20 mL). The combined organic layer was washed by 5% aqueous NaHCO₃ (20 mL), saturated aqueous brine (3×20 mL), and dried over Na₂SO₄. The solvent was concentrated in vacuum to give an oil product 129 (3.0 g, yield 60%, purity 80%)

Step 4: Synthesis of (S)-tert-butyl (3-(4-(3-(2,5-dimethoxy-3,4,6-trimethylphenyl) propoxy)phenyl)-1-(isopropylamino)-1-oxopropan-2-yl)carbamate (124)

To a solution of compound 123 (0.5 g, 1.56 mmol) in DMSO (10 mL) was added K₂CO₃ (650 mg, 4.68 mmol) and 3-(2,5-dimethoxy-3,4,6-trimethylphenyl)propyl 4-methylbenzenesulfonate (129, 733 mg, 1.9 mmol) at 100° C. The reaction mixture was stirred for 16 h, and then water was added the mixture and extracted by EA (2×10 mL). The combine layer was washed with aqueous brine (20 mL), dried over Na₂SO₄ and concentrated in vacuum. The residue was subjected to column chromatography on silica gel (petroleum ether/EtOAc=10:1 to 2:1) to give the titled product 124 (350 mg, 41%) as an oil. MS (ESI): [M+H⁺]=543.2.

Step 5: Synthesis of (S)-2-amino-3-(4-(3-(2,5-dimethoxy-3,4,6-trimethylphenyl) propoxy)phenyl)-N-isopropylpropanamide (125)

A solution of compound 124 (350 mg, 0.64 mmol) in HCl in MeOH (10 mL, 4 M) was stirred at 25° C. for 2 h and then the solvent was concentrated in vacuum to give the titled product 125 (283 mg, 100%) as an oil. MS (ESI): [M+H⁺]=443.2.

Step 6: Synthesis of (S)-tert-butyl 2-(((S)-3-(4-(3-(2,5-dimethoxy-3,4,6-trimethylphenyl)propoxy)phenyl)-1-(isopropylamino)-1-oxopropan-2-yl) carbamoyl)pyrrolidine-1-carboxylate (126)

To the solution of compound 125 (283 mg, 0.64 mmol) and (S)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid (70) (165 mg, 0.77 mmol) in DCM (10 mL) at 0° C. was added DIEA (248 mg, 1.92 mmol), and T₃P (50% in EA, 9.4 g, 14.9 mmol) with dropwise, and the reaction mixture was allowed to warm to 25° C. and stirred for 16 h. The reaction mixture was washed with 5% aqueous Na₂CO₃ (10 mL) and water (40 mL), and the organic layer was dried over MgSO₄ and concentrated in vacuum to obtain a solid product 126 (290 mg, 71%). MS (ESI): [M+H⁺]=640.3.

Step 7: Synthesis of (S)-tert-butyl 2-(((S)-1-(isopropylamino)-1-oxo-3-(4-(3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propoxy)phenyl)propan-2-yl) carbamoyl) pyrrolidine-1-carboxylate (127)

A solution of compound 126 (290 mg, 0.45 mmol) in THF (8 mL) was diluted with water (2 mL), and an excess solution of ceric ammonium nitrate (CAN) (543 mg, 0.99 mmol) in 5 mL water was added at 0° C. The mixture was stirred at room temperature for 2 h. The progress of the reaction was monitored by TLC. After completion, THF was removed under a vacuum and the crude mixture was extracted with three portions of EA (3×20 mL). The combined organic layer was washed with aqueous brine solution (20 mL), then dried over anhydrous Na₂SO₄ and concentrated in vacuum. The crude products were purified by a silica-gel column chromatography (petroleum ether/EtOAc=5:1 to 2:1) to give the titled product 127 (160 mg, 58%) as a yellow oil. MS (ESI): [M+H⁺]=610.2.

Step 8: Synthesis of (S)—N-((S)-1-(isopropylamino)-1-oxo-3-(4-(3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propoxy)phenyl)propan-2-yl)pyrrolidine-2-carboxamide (128)

A solution of compound 127 (160 mg, 0.26 mmol) in HCl in MeOH (10 mL, 4 M) was stirred at 25° C. for 2 h and then concentrated to give the titled product 128 (132 mg, 100%) as an oil. MS (ESI): [M+H⁺]=510.1.

Step 9: Synthesis of tert-butyl ((4S,7S,E)-7-benzyl-8-((S)-2-(((S)-1-(isopropylamino)-1-oxo-3-(4-(3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propoxy)phenyl)propan-2-yl)carbamoyl)pyrrolidin-1-yl)-2-methyl-8-oxooct-5-en-4-yl)carbamate (X)

To the solution of compound 128 (132 mg, 0.26 mmol) and (2S,5S,E)-2-benzyl-5-((tert-butoxycarbonyl)amino)-7-methyloct-3-enoic acid (70, 113 mg, 0.31 mmol) in DCM (10 mL) at 0° C. was added DIEA (74 mg, 0.57 mmol), and T₃P (50% in EA, 248 mg, 0.39 mmol), and the reaction mixture was allowed to warm to 25° C. and stirred for 16 h. The reaction mixture was washed with 5% Na₂CO₃ (10 mL) and water (20 mL), and the organic layer was dried over MgSO₄ and concentrated in vacuum. The residues were purified by pre-HPLC (Durashell C18(10 μm, 30×250 mm), 0.1% NH₃.H₂O in eluant) to obtain a yellow solid product X (42 mg, 19%). MS (ESI): [M+H⁺]=853.4. ¹H NMR (400 MHz, CDCl₃) δ 7.24-7.06 (m, 7H), 6.76-6.62 (m, 2H), 5.669-5.31 (m, 3H), 4.43-4.39 (m, 2H), 4.15-3.88 (s, 3H), 3.45-3.34 (m, 2H), 3.14-3.09 (m, 2H), 2.95-2.90 (m, 2H), 2.72-2.63 (m, 2H), 2.00-0.87 (m, 43H).

Example 8: tert-butyl N-[(4S,5E,7S)-7-benzyl-2-methyl-8-[(2S)-2-{[(1S)-2-methyl-1-{[[(1S)-1-[(propan-2-yl)carbamoyl]-4-({3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propoxy]carbonyl}amino)butyl]carbamoyl}propyl] carbamoyl}pyrrolidin-1-yl]-8-oxooct-5-en-4-yl]carbamate (XI)

Step 1: Synthesis of (S)-tert-butyl (3-(2,5-dimethoxy-3,4,6-trimethylphenyl)propyl) (5-(isopropylamino)-5-oxopentane-1,4-diyl)dicarbamate (133)

To a solution of 52 (500 mg, 1.01 mmol) in DCM (10 mL) was added DBU (768 mg, 5.05 mmol) at 0° C., the reaction mixture was stirred for 30 min and allowed to warm to 25° C. and then compound 110 (364 mg, 1.21 mmol) and DIEA (291 mg, 1.52 mmol) were added. After 30 min later, water (20 mL) was added to the mixture, and extracted by DCM (2×10 mL). The combined organic layer was washed with aqueous brine (20 mL), dried over Na₂SO₄ and concentrated in vacuum. The crude products were purified by a silica-gel column chromatography (petroleum ether/EtOAc=5:1 to 2:1) to give the titled product 133 (420 mg, 77%) as a yellow solid. MS (ESI): [M+H⁺]=538.2.

Step 2: Synthesis of (S)-3-(2,5-dimethoxy-3,4,6-trimethylphenyl)propyl (4-amino-5-(isopropylamino)-5-oxopentyl)carbamate (134)

A solution of compound 133 (420 mg, 0.78 mmol) in HCl in MeOH (10 mL, 4 M) was stirred at 25° C. for 2 h and then the solvent was concentrated in vacuum to give the titled product 134 (341 mg, 100%) as an oil. MS (ESI): [M+H⁺]=438.2.

Step 3: Synthesis of (S)-3-(2,5-dimethoxy-3,4,6-trimethylphenyl)propyl (4-amino-5-(isopropylamino)-5-oxopentyl)carbamate (135)

To the solution of compound 134 (420 mg, 0.78 mmol) and (S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanoic acid (203 mg, 0.93 mmol) in DCM (10 mL) at 0° C. was added DIEA (221 mg, 1.72 mmol), and T₃P (50% in EA, 744 mg, 1.17 mmol), and the reaction mixture was allowed to warm to 25° C. and stirred for 16 h. The reaction mixture was washed with 5% aqueous Na₂CO₃ (10 mL) and water (20 mL), and the organic layer was dried over MgSO₄ and concentrated in vacuum to obtain a solid product 135 (420 g, 85%). MS (ESI): [M+H⁺]=637.2.

Step 4: Synthesis of 3-(2,5-dimethoxy-3,4,6-trimethylphenyl)propyl ((S)-4-((S)-2-amino-3-methylbutanamido)-5-(isopropylamino)-5-oxopentyl)carbamate (136)

A solution of 135 (420 mg, 0.66 mmol) in HCl in MeOH (10 mL, 4 M) was stirred at 25° C. for 2 h and then concentrated in vacuum to give the titled compound 136 (354 mg, 100%) as an oil. MS (ESI): [M+H⁺]=537.3.

Step 5: Synthesis of (S)-tert-butyl 2-(((S)-1-(((S)-5-(((3-(2,5-dimethoxy-3,4,6-trimethylphenyl)propoxy)carbonyl)amino)-1-(isopropylamino)-1-oxopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (137)

To the solution of compound 136 (354 mg, 0.66 mmol) and (S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanoic acid (212 mg, 0.99 mmol) in DCM (10 mL) at 0° C. was added DIEA (187 mg, 1.45 mmol), and T₃P (50% in EA, 630 mg, 0.99 mmol), and the reaction mixture was allowed to warm to 25° C. and stirred for 16 h. The reaction mixture was washed with 5% aqueous Na₂CO₃ (10 mL) and water (20 mL), and the organic layer was dried over MgSO₄ and concentrated in vacuum to obtain a solid product 137 (350 mg, 72%). MS (ESI): [M+H⁺]=734.3.

Step 6: Synthesis of (2S)-tert-butyl 2-(((2S)-1-(((2S)-1-(isopropylamino)-1-oxo-5-(((3-(2,4,5-trimethyl-3,6-dioxocyclohex-1-en-1-yl)propoxy)carbonyl)amino) pentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (138)

A solution of compound 138 (350 mg, 0.48 mmol) in THF (8 mL) was diluted with water (2 mL), and an excess solution of ceric ammonium nitrate (CAN) (580 mg, 1.05 mmol) in 5 mL water was added at 0° C. The mixture was stirred at room temperature for 2 h. The progress of the reaction was monitored by TLC. After completion, THF solvent was removed under a vacuum and the crude mixture was extracted with three portions of EA (3×20 mL). The organic extracts were washed with aqueous brine (2×20 mL), then dried over anhydrous Na₂SO₄ and concentrated in vacuum. The crude products were purified by a silica-gel column chromatography (petroleum ether/EtOAc=5:1 to 2:1) to give the titled product 138 (190 mg, 56%) as a yellow oil. MS (ESI): [M+H]=704.4. ¹H NMR (400 MHz, CDCl₃) δ 7.13-6.56 (m, 3H), 5.03 (s, 1H), 4.44-3.99 (m, 6H), 3.75-3.44 (m, 5H), 2.54-0.88 (m, 44H).

Step 7: Synthesis of 3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propyl ((S)-5-(isopropylamino)-4-((S)-3-methyl-2-((S)-pyrrolidine-2-carboxamido) butanamido)-5-oxopentyl)carbamate (139)

A solution of compound 138 (190 mg, 0.27 mmol) in HCl in MeOH (10 mL, 4 M) was stirred at 25° C. for 2 h and then concentrated in vacuum to give the titled product 138 (162 mg, 100%) as an oil. MS (ESI): [M+H]=604.3.

Step 8: Synthesis of tert-butyl N-[(4S,5E,7S)-7-benzyl-2-methyl-8-[(2S)-2-{[(1S)-2-methyl-1-{[(1S)-1-[(propan-2-yl)carbamoyl]-4-({[3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propoxy]carbonyl}amino)butyl]carbamoyl}propyl] carbamoyl}pyrrolidin-1-yl]-8-oxooct-5-en-4-yl]carbamate (XI)

To the solution of compound 139 (162 mg, 0.27 mmol) and (2S,5S,E)-2-benzyl-5-((tert-butoxycarbonyl)amino)-7-methyloct-3-enoic acid (70, 113 mg, 0.31 mmol) in DCM (10 mL) at 0° C. was added DIEA (74 mg, 0.57 mmol), T₃P (50% in EtOAc, 248 mg, 0.39 mmol) slowly, and the reaction mixture was allowed to warm to 25° C. and stirred for 16 h. The reaction mixture was washed with 5% aqueous Na₂CO₃ (10 mL) and water (20 mL), and the organic layer was dried over MgSO₄ and concentrated in vacuum. The residues were purified by prep-HPLC (Durashell C18(10 μm, 30×250 mm), 0.1% NH₃.H₂O in eluant) to obtain a yellow solid product XI (45 mg, 18%). MS (ESI): [M+H]=947.6. ¹H NMR (400 MHz, CDCl₃) δ=7.24-7.13 (m, 5H), 6.79-6.33 (m, 2H), 5.63-5.27 (m, 3H), 4.79-4.03 (m, 6H), 3.57-2.50 (m, 7H), 2.00-0.85 (m, 55H).

Example 9: 3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propyl N-[(4S)-4-{[(2S)-1-[(2S,3E,5S)-2-benzyl-5-{[(tert-butoxy)carbonyl]amino}-7-methyloct-3-enoyl]pyrrolidin-2-yl]formamido}-4-(cyclopropylcarbamoyl)butyl] carbamate (XII)

Step 1: Synthesis of (S)-(9H-fluoren-9-yl)methyl tert-butyl (5-(cyclopropylamino)-5-oxopentane-1,4-diyl)dicarbamate (142)

To the solution of compound 141 (0.5 g, 1.1 mmol) and cyclopropylamine (94 g, 1.7 mmol) in DCM (10 mL) at 0° C. was added DIEA (220 mg, 1.7 mmol), T₃P (50% in EA, 1.1 g, 1.7 mmol), and the reaction mixture was allowed to warm to 25° C. and stirred for 16 h. The reaction mixture was washed with 5% aqueous Na₂CO₃ and water, and the organic layer was dried over MgSO₄ and concentrated in vacuum to obtain a solid product 142 (480 mg, 89%). MS (ESI): [M+H⁺]=494.2.

Step 2: Synthesis of (S)-(9H-fluoren-9-yl)methyl (4-amino-5-(cyclopropylamino)-5-oxopentyl)carbamate (143)

A solution of compound 142 (480 mg, 0.98 mmol) in HCl in MeOH (10 mL, 4 M) was stirred at 25° C. for 2 h and then concentrated in vacuum to give the product 143 (385 mg, 100%) as an oil. MS (ESI): [M+H⁺]=394.0.

Step 3: Synthesis of (S)-tert-butyl 2-(((S)-5-((((9H-fluoren-9-yl)methoxy) carbonyl)amino)-1-(cyclopropylamino)-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (144)

To the solution of compound 143 (385 mg, 0.98 mmol) and (S)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid (316 mg, 1.47 mmol) in DCM (10 mL) at 0° C. was added DIEA (278 mg, 2.2 mmol), T₃P (50% in EA, 934 mg, 1.47 mmol), and the reaction mixture was allowed to warm to 25° C. and stirred for 16 h. The reaction mixture was washed with 5% aqueous Na₂CO₃ (20 mL), and the organic layer was dried over MgSO₄ and concentrated in vacuum to obtain a solid product 144 (390 mg, 68%). MS (ESI): [M+H⁺]=591.2.

Step 4: Synthesis of (S)-tert-butyl 2-(((S)-1-(cyclopropylamino)-5-(((3-(2,5-dimethoxy-3,4,6-trimethylphenyl)propoxy)carbonyl)amino)-1-oxopentan-2-yl) carbamoyl)pyrrolidine-1-carboxylate (146)

To a solution of compound 144 (390 mg, 0.67 mmol) in DCM (10 mL) was added DBU (510 mg, 3.35 mmol) at 0° C. The reaction mixture was allowed to warm to 25° C. and stirred for 30 min and then added compound 110 (240 mg, 0.8 mmol) and DIEA (110 mg, 0.8 mmol). Water was added the mixture, and the mixture was extracted by DCM (2×10 mL). The combined layer was washed with aqueous brine (3×10 mL), dried over Na₂SO₄ and concentrated in vacuum. The residue was purified via column (silica gel, PE/EA=3:1 to 1:1) to give the product 146 (340 mg, 75%) as a yellow solid. MS (ESI): [M+H⁺]=633.4.

Step 5: Synthesis of (S)-tert-butyl 2-(((S)-1-(cyclopropylamino)-1-oxo-5-(((3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propoxy)carbonyl)amino) pentan-2-yl) carbamoyl)pyrrolidine-1-carboxylate (147)

A solution of compound 146 (340 mg, 0.5 mmol) in THF (8 mL) was diluted with water (2 mL), and an excess solution of ceric ammonium nitrate (CAN) (602 mg, 1.1 mmol) in 5 mL water was added at 0° C. The mixture was stirred at room temperature for 2 h. The progress of the reaction was monitored by TLC. After completion, THF was removed under a vacuum and the crude mixture was extracted with three portions of EA (3×20 mL). The organic extracts were washed with aqueous brine (3×20 mL), then dried over anhydrous Na₂SO₄ and concentrated in vacuum. The crude products were purified by a silica-gel column chromatography on silica gel (PE/EA=3:1 to 1:1) to give the product 147 (180 mg, 44%) as a yellow oil. MS (ESI): [M+H⁺]=603.2. ¹H NMR (400 MHz, CDCl₃) δ 7.26-6.77 (m, 2H), 4.93 (s, 1H), 4.44-4.10 (m, 4H), 3.66-3.63 (m, 6H), 3.49-3.3.45 (m, 4H), 2.68-2.64 (m, 3H), 2.20-1.25 (m, 28H), 0.72-0.53 (m, 4H).

Step 6: Synthesis of 3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propyl ((S)-5-(cyclopropylamino)-5-oxo-4-((S)-pyrrolidine-2-carboxamido)pentyl) carbamate (148)

A solution of compound 147 (180 mg, 0.22 mmol) in HCl in MeOH (10 mL, 4 M) was stirred at 25° C. for 2 h and then the solvent was concentrated in vacuum to give the product 148 (110 mg, 100%) as an oil. MS (ESI): [M+H⁺]=503.2.

Step 7: Synthesis of 3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)propyl N-[(4S)-4-{[(2S)-1-[(2S,3E,5S)-2-benzyl-5-{[(tert-butoxy)carbonyl]amino}-7-methyloct-3-enoyl]pyrrolidin-2-yl]formamido}-4-(cyclopropylcarbamoyl)butyl] carbamate (XII)

To the solution of compound 148 (110 mg, 0.22 mmol) and (2S,5S,E)-2-benzyl-5-((tert-butoxycarbonyl)amino)-7-methyloct-3-enoic acid (70, 95 mg, 0.26 mmol) in DCM (10 mL) at 0° C. was added DIEA (62 mg, 0.48 mmol), T₃P (50% in EA, 210 mg, 0.33 mmol), and the reaction mixture was allowed to warm to 25° C. and stirred for 16 h. The reaction mixture was washed with 5% aqueous Na₂CO₃ (20 mL) and water (20 mL), and the organic layer was dried over MgSO₄ and concentrated in vacuum. The residues were purified by prep-HPLC (Durashell C18(10 μm, 30×250 mm), 0.1% NH₃.H₂O in eluant) to obtain a yellow solid product XII (15 mg, 8%). MS (ESI): [M+H⁺]=846.5. ¹H NMR (400 MHz, CDCl₃) δ 7.25-7.09 (m, 8H), 5.61-5.27 (m, 3H), 4.33 (s, 2H), 4.02 (s, 2H), 3.60-2.75 (m, 12H), 1.98-0.58 (m, 39H).

Example 10 Primary Screening by Ferroptosis Assay

Cell Lines and Media: HT-1080 (fibrosarcoma) cells were obtained from American Type Culture Collection. Cells were grown in EMEM media, 10% Heat-Inactivated FBS, and penicillin-streptomycin mix (Invitrogen). Cells were maintained at 37° C. and 5% CO₂ in a tissue culture incubator.

Cell Viability Assay. Seed 10,000 cell/well of HT-1080 cells with 195 μL growth media in black and clear bottom 96-well plates (Cat #Corning3904) and allow cells to adhere overnight. The next day, dilute test compound by 2-fold dilution into 12 points with medium containing 400 μM Erastin. Transfer 5 μL of compounds solution into growth medium in triplicated wells. 30 hours later, add Cell Titer-Glo® Luminescent cell viability assay reagent (Cat #, Promega G8081) to measure HT-1080 cell viability according to the manufacture protocol. Briefly, aspirate 100 μL medium, and add 50 μL CellTiter-Glo® Luminescent reagent to each well and incubated for 30 minutes at room temperature to stabilize the Luminescent signal. Seal the plates and centrifuged for 1 minute at 1,000 rpm to remove bubbles. Shake the plates for 1 minute on an orbital shaker. Read the plate to detect Luminescent signal with EnSpire Multimode Plate Reader (PerkinElmer). The remaining activity % is used to evaluate Erastin induced ferroptosis in HT-1080 cells and expressed as the following formula:

% Remaining Activity=100×[(Sample−Background_(avg))/(Vehicle−Background_(avg))]

Sample: Readout from the test compound

Vehicle: Readout from the vehicle sample

Background: Readout from the Erastin only treatment

Dose response curve is graphed by using the non-linear regression analysis (formula equation 201) in XLFit (Excel add-in software) for the average % remaining activity from triplicates, and the EC50 values is then calculated. In this anti-ferroptosis assay, ferrostatin-1, and XJB-5-131 are included as reference. The acceptance criteria were set based on historical result, mean±2.58*SD in logarithmic scale, meanwhile Z′>0.5.

TABLE 1 Summary of Example Compounds Inhibition for Ferroptosis in HT-1080 Cell. Example ID EC50/nM I 118.98 II 32.96 III 34.57 IV 24.38 V 196.06 VI 39.06 VII 80.55 VIII >6000 IX 49.29 X 56.86 XI 47.01 XII 148.72

Example 11

Apoptosis Assay

Cell Lines and Media. 3T3-L1 cells were obtained from American Type Culture Collection. Cells were grown in DMEM completed media, 10% FBS, and penicillin-streptomycin mix (Invitrogen). Cells were maintained at 37° C. and 5% CO₂ in a tissue culture incubator.

Apoptosis Assay. Promega's Caspase-Glo®3/7 assay uses a luminogenic substrate containing the DEVD sequence, which has been shown to be selective for caspase-3 and -7. We used this assay to screen our compounds for apoptosis inhibition. Briefly, 40 μL of 3000 3T3-L1 cells were seeded per well in 384 well plates, Cells were allowed to adhere overnight. In the next day, prepare the Actinomycin D as the apoptosis inducing agent into 100 μM DMSO stock. Prepare the test compounds into 10 mM DMSO stock. Serial dilute the compound into 10 points by 3-fold dilution with DMSO. Pre-treat the 3T3-L1 cells with test compound by transfer 40 nL of stock of serial concentration in triplicate wells using acoustic fluid transfer, incubate for 2 hours. Then transfer 40 nL of 100 μM of Actinomycin D (final concentration is 100 nM) into designated wells using acoustic fluid transfer. Incubate the assay plates at 37° C., 5% CO₂, 95% humidity incubator for 48 hours. For caspase assay, add 20 μL/well of Caspase-Glo® 3/7 reagent (Promega) into test wells. Incubate for 30 minutes at room temperature to stabilize the luminescence signal. Read the plate with EnSpire Multimode Plate Reader (PerkinElmer). Calculate the EC50 value using formula equation 201: y=(A+((B−A)/(1+((x/C){circumflex over ( )}D)))) from the XLFit (Excel add-in software), where

A: Average mean of vehicle samples

B: Average mean of cells treated with 100 nM AcD

C: EC50

D: HillSlope

Z′ factor was used to describe the screening assay performance. The Z′ factor value was >0.5 in all the test plates which indicated the assay was qualified and the IC50 value was reliable for all tested compounds.

TABLE 2 Summary of Example Compounds Inhibition for Apoptosis in 3T3-L1 Cell induced by Actinomycin D. Example Compounds EC50/μM I 1.152 II 0.808 II 1.566 IV 0.702 V 2.745 VI 7.317

Example 12

Lipid ROS Assay to Evaluate Compound

DCFDA-Cellular Reactive Oxygen Species Detection Assay Kit from Abcam(ab113851) was used to detect cellular (lipid) ROS in HT-1080 cells induced by Erastin.

DCFDA-Cellular ROS Detection Assay Kit (ab113851) uses the cell permeant reagent 2′,7′-dichlorofluorescin diacetate (DCFDA), a fluorogenic dye that measures hydroxyl, peroxyl and other reactive oxygen species (ROS) activity within the cell. After diffusion into the cell, DCFDA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into 2′, 7′-dichlorofluorescein (DCF). DCF is a highly fluorescent compound which can be detected by fluorescence spectroscopy with maximum excitation and emission spectra of 495 nm and 529 nm respectively

Lipid ROS Assay. 25000 HT-1080 cells were seeded into black and clear bottom 96-well plates, let the HT-1080 cells adhere overnight. Next day, treat the cells with cytotoxic agent (Erastin, 50 μM) and incubate with test compounds (10 points, 3-fold series dilution) for 24 hours. Wash cells in 1×PBS and stain cells with DCFDA reagent for 30-45 minutes at 37° C. incubator according to the manufacture protocol (Ab113851). Measure fluorescence (Ex/Em=485/535 nM) in the assay plates using EnSpire Multimode Plate Reader (PerkinElmer). Determine the ROS change as a percentage of control after background subtraction. Dose response curve was graphed by using the non-linear regression analysis in XLFit software for the average % ROS change from duplicates, and the EC50 values were calculated. In addition, lipid ROS can also be measured by flow cytometry using the FL1 channel (green fluorescence), or by fluorescence microscopy. The fluorescence can be detected by using excitation and emission wavelengths that are appropriate for green fluorescence.

Example compounds were used to test the ability of inhibition on the lipid ROS assay, most of examples have the ability inhibition on lipid ROS with IC50 less than 1 mM.

Example 13a Ischemia Reperfusion (IR) Induced Acute Kidney Injury (AKI) Animal Model

Mice were anesthetized with a 5% chloral hydrate (400 mg/kg 5% chloral hydrate). Bilateral renal ischemia was induced by the application of non-traumatic micro-vascular clamps around both left and right renal pedicles. Ischemia was confirmed by blanching of the kidneys. After 30-minute ischemia, the clamps were removed and reperfusion was confirmed visually. Sham-operated animals were not subjected to ischemia. During the ischemic interval, the animals were kept hydrated with normal saline instilled intraperitoneally and were kept on warm heating pads to maintain body temperature.

Animals were randomly assigned to the following groups: Sham operated, IR with saline, or IR with test article (5.0 mg/kg, 15 mg/kg). Treatment was administered subcutaneously 2 h before onset of ischemia, at the onset of reperfusion, and at 2 hours after reperfusion. Serum and urine samples were collected at 24 hours and stored at −20° C. until analysis. Kidneys were harvested at different times after onset of reperfusion 24 hours for assessment of oxidative markers, and histopathology by light and electron microscopy.

The effect of example compounds not only decrease the creatinine and BUN to normal function level, but also shown protection on the kidney histological damage in renal IR.

Example 13b Folic Acid-Induced Acute Kidney Injury (AKI) Animal Model

Mice were divided into four groups, sham control, folic acid treatment group that contains the following sub groups: article 1 treatment group, article 2 treatment group, and DMSO (vehicle control) group. Mice were dosed intraperitoneally with article 1, article 2, or DMSO (vehicle) 30 minutes before FA injection. The three folic acid treatment groups were administered a single intraperitoneal injection of FA (Sigma-Aldrich, St. Louis, Mo.) of 250 mg/kg in 0.3 mol/L sodium bicarbonate Animals were sacrificed 48 hours after treatment.

Plasma samples were collected at the time of euthanasia. Kidneys were perfused in situ with cold saline before removal. One kidney was snap frozen in liquid nitrogen for RNA and protein studies, and the other was fixed and paraffin embedded to archive for histopathology analysis.

The effect of example compounds showed the efficacy in the FA-induced AKI model by decreasing the creatinine and BUN to normal function and the kidney with little damage compared with vehicle control. From molecular signal pathway, example compound can effectively decrease the expression of RIPK3 and MLKL, which is associated with renal inflammation.

Finally, it should be noted that there are other ways to practice the invention. Accordingly, embodiments of the present invention is to be described as examples, but the present invention is not limited to the contents described, further modifications may be made within the scope of the present invention or the equivalents added in the claims.

All publications or patents cited herein are incorporated by reference in this invention.

Reference throughout this specification to “an embodiment”, “some embodiments”, “one embodiment”, “another example”, “an example”, “a specific example” or “some examples” means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, the appearances of the phrases such as “in some embodiments,” “in one embodiment”, “in an embodiment”, “in another example, “in an example,” “in a specific example,” or “in some examples,” in various places throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

Although explanatory embodiments have been shown and described, it would be appreciated by those skilled in the art that the above embodiments cannot be construed to limit the present disclosure, and changes, alternatives, and modifications can be made in the embodiments without departing from spirit, principles and scope of the present disclosure. 

What is claimed is:
 1. A method of treating ischemia in a patient comprising administering to a patient in need of such treatment a therapeutically acceptable amount a compound of Formula (I)a or Formula (I)b, a pharmaceutically acceptable salt thereof, or an individual enantiomer or diastereomer thereof:

wherein, z is 0 or 1; L is absent; or L is selected from C₁-C₁₀ alkyl, C₁-C₁₀ alkoxyl, C₃₋₇ cycloalkyl, C₁-C₁₀ alkylcycloalkyl, heterocycloalkyl, C₁-C₁₀ alkylheterocycloalkyl, C₅₋₁₀ aryl, C₁-C₁₀ alkylaryl, C₅₋₁₀ heteroaryl, and C₁-C₁₀ alkylheteroaryl; Q is selected from:

wherein, n is an integer selected from 0 to 10; each of R¹ and R² is independently selected from hydrogen and CH₃; each of R³ and R⁴ is independently selected from hydrogen and C₁-C₅ alkyl; and each W is independently absent or is selected from O and S; or i) when W is absent, then R³ and R⁴, form the following structure:

 or ii) when W is O, then OR³ and OR⁴, together form the following structure:

 and PEP is a peptidyl moiety having the following structure:

wherein, each asterisk (*) independently denotes a chiral center in either an (S) or (R) configuration; m is an integer selected from 0 to 10; X¹ is selected from hydrogen, —C(═O)—O—R_(m), —S(O)—O—R_(m), —S(O₂)—O—R_(m), —S(O₂)—N—(R_(m))₂ and —C(═O)—N—(R_(m))₂, where R_(m) is C₁-C₆ alkyl, C₁-C₆ alkoxyl, C₁-C₆ alkenyl, C₁-C₆ haloalkyl, C₃-C₇ cycloalkyl, C₁-C₆ alkylcycloalkyl, C₃-C₇ heterocycloalkyl, C₁-C₆ alkylheterocycloalkyl, C₅-C₁₀ aryl, C₁-C₆ alkylaryl, C₅-C₁₀ heteroaryl, and C₁-C₆ alkylheteroaryl; each of X², X³ and X⁴ is independently selected from C₁-C₁₀ alkyl, C₁-C₁₀ alkenyl, C₅-C₆ aryl, C₁-C₆ alkylaryl, heteroaryl, and C₁-C₆ alkylheteroaryl; A is absent, or A is selected from Ala, Leu, Ile, Phe, Met, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Gln, His, Lys, Arg, Asp, Glu, and Val, each of which can be in either a D or L configuration; wherein the amino residue is either unprotected, or protected by a protecting group selected from Cbz and Fmoc; and G is absent, or G is selected from —O—, —S—, —NH—, —NH—C(═O)—O—, —O—C(═O)—NH—, —NH—C(═O)—NH—, —NHSO₂—, —SO—, —SO₂—, and —NHC(═O)—.
 2. The method of claim 1, wherein L is C₁-C₁₀ alkyl.
 3. The method of claim 1, wherein L is selected from C₅-C₁₀ aryl and C₁-C₁₀ alkylaryl.
 4. The method of claim 1, wherein L is selected from C₅-C₁₀ heteroaryl, C₁-C₁₀ alkylaryl, C₁-C₁₀ alkylheteroaryl, and pyridine.
 5. The method of claim 1, wherein G is absent or G is selected from —NHC(═O)—, —NHC(═O)—O—, and —NHSO₂—.
 6. The method of claim 1, wherein Q has the structure:

wherein, each W is independently absent; n is an integer from 1 to 3; R¹ is selected from hydrogen and methyl; and each R³ is independently C₁₋₃ alkyl.
 7. The compound according to claim 1, wherein PEP has the structure:

wherein, each asterisk (*) independently denotes a chiral center that can be either in an (S) or (R) configuration; X¹ is selected from —C(═O)—O—R_(m), —S(O)—O—R_(m), —S(O₂)—O—R_(m), —S(O₂)—N—(R_(m))₂, and —C(═O)—N—(R_(m))₂, where R_(m) is selected from C₁-C₆ alkyl, C₁-C₆ alkoxyl, C₁-C₆ alkenyl, C₁-C₆ haloalkyl, C₃-C₇ cycloalkyl, C₁-C₆ alkylcycloalkyl, C₃-C₇ heterocycloalkyl, C₁-C₆ alkylheterocycloalkyl, C₅-C₁₀ aryl, C₁-C₆ alkylaryl, C₅-C₁₀ heteroaryl, and C₁-C₆ alkylheteroaryl; each of X₂, X₃ and X₄ is independently selected from C₁-C₁₀ alkyl, C₁-C₁₀ alkenyl, C₅-C₆ aryl, C₁-C₆ alkylaryl, C₅-C₆ heteroaryl, and C₁-C₆ alkylheteroaryl; and A is absent or A is selected from Ala, Leu, Ile, Phe, Met, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Gln, His, Lys, Arg, Asp, Glu, and Val, each of which can be in either a D or L configuration; wherein the amino residue is either unprotected, or protected by a protecting group selected from Cbz and Fmoc.
 8. The method of claim 7, wherein A is absent or A is valine.
 9. The method of claim 7, wherein, X¹ is absent, or X¹ is selected from Et-O—C(═O)—, i-Pr—O—C(═O)—, t-Bu-O—C(═O)—, and cyclopropyl-CH₂—O—C(═O)—; X² is selected from i-propyl-CH₂—, cyclopropyl-CH₂—, cyclopentyl-CH₂—, and adamantyl-CH₂—; X³ is selected from Ph-CH₂— and Pyr-CH₂—; and X⁴ is selected from i-propyl-CH₂—, cyclopropyl-CH₂—, and cyclopentyl-CH₂—.
 10. The method of claim 7, wherein, A is absent; X¹ is selected from t-Bu-O—C(═O)—, cyclopropyl-CH₂—O—C(═O)—, and ethyl-CH₂—O—C(═O)—; X² is i-propyl-CH₂—; X³ is Ph-CH₂—; and X⁴ is i-propyl-CH₂—.
 11. The method of claim 7, wherein, A is absent; X¹ is t-Bu-O—C(═O)—; X² is selected from i-propyl-CH₂— and adamantyl-CH₂—; X³ is Ph-CH₂—; and X⁴ is selected from cyclopropyl-CH₂—, cyclopentyl-CH₂—, and i-propyl-CH₂—.
 12. The method of claim 7, wherein. A is valine; X¹ is selected from t-Bu-O—C(═O)— and cyclopropyl-CH₂—O—C(═O)—; X² is selected from i-propyl-CH₂— and adamantyl-CH₂—; X₃ is Ph-CH₂—; and X⁴ is selected from i-propyl-CH₂—, cyclopropyl-CH₂—, and cyclopentyl-CH₂—.
 13. The method of claim 1, wherein the compound is selected from:

a pharmaceutically acceptable of any of the foregoing.
 14. The method of claim 1, wherein the administering comprises administering a pharmaceutical composition comprising the compound of Formula (I)a, Formula (I)b, a combination thereof, a pharmaceutically acceptable salt thereof, or an individual enantiomer or diastereomer thereof.
 15. The method of claim 1, wherein the ischemia is associated with acute kidney injury.
 16. The method of claim 1, wherein the ischemia is associated with stroke.
 17. The method of claim 1, wherein the ischemia is associated with atherosclerosis.
 18. The method of claim 1, wherein the ischemia is associated with hypertensive cardiomyopathy.
 19. The method of claim 1, wherein the ischemia is associated with congestive heart failure. 